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 7^th 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.     

Biochemical properties of different entomopathogenic fungi and their virulence against Chilo suppressalis (Lepidoptera: Crambidae) larvae

We determined the virulence of Beauveria bassiana, Metarhizium anisopliae, Isaria fumosoroseus and Lecanicilium lecanii against larvae of Chilo suppressalis Walker by bioassay and evaluated several enzymatic and non-enzymatic components. LC₅₀ values of the entomopathogenic fungi revealed 90, 32, 45,000, 4600, 42,000 and 1,540,000 spores/larva for isolates BB1–BB3 of B. bassiana, I. fumosoroseus, M. anisopliae and L. lecanii, respectively. Isolate BB3 and I. fumosoroseus had the highest amounts of total protein and hydrophobin and isolates BB3 and M. anisopliae showed the highest activities of lipases and chitinases. In case of proteases, the highest activities were observed for Pr1 of BB1 and Pr2 of L. lecanii. The highest general esterase activities were obtained in I. fumosoroseus and BB1 when 1-naphtyl acetate and 2-naphtyl acetate were used as substrates. The highest activity of glutathione S-transferase (GST) was observed in I. fumosoroseus by using both reagents but BB1 demonstrated the highest activities of alkaline phosphatase and acid phosphatase. Clustering of the fungi using biochemical enzymes revealed BB2 and BB3 as a separate group of entomopathogenic fungi. In another group, I. fumosoroseus and L. lecanii had the most similarity and were separated from BB1 and M. anisopliae. The fungi exhibited different virulence on larvae of C. suppressalis by producing adhering protein and extracellular enzymes. Overall, results of the bioassays and clustering based on enzymatic activities revealed that isolate BB2 was the most effective fungus against larvae of C. suppressalis.     

Time-dose-mortality modelling and virulence indices for six strains of Verticillium lecanii against sweetpotato whitefly, Bemisia tabaci (Gennadius)

Bioassays of six strains of Verticillium lecanii (Zimmermann) Viégas were conducted with the sweet potato whitefly, Bemisia tabaci. For inoculation, batches of third‐instar whitefly nymphs on sweetpotato seedlings were immersed in conidial suspensions of five dosages at concentrations from 10³ to 10⁷ conidia/ml. Each dosage was used to inoculate 150–250 nymphs. The nymphs were maintained at 25°C, under 95% RH, photo phase of 16 : 8 (L : D) and observed daily for mortality. The resulting data were analysed over 8 days by a complementary log‐log (CLL) time‐dose‐mortality model, based on the Hosmer–Lemeshow test, analysing the time‐dose trends for five concentrations of six strains of V. lecanii simultaneously. The parameters from the model were used to estimate the virulence indices (the values of LC₅₀) of six strains against B. tabaci. Based on the time‐dose‐mortality relationships fitted and the virulence indices, the virulence of the six strains of V. lecanii for B. tabai was compared. Results indicated that the strain Vl6063 imported from Canada and the domestic strains V3450 and Vp28 derived from B. tabaci and a scale insect, respectively, were more virulent than the others with LC₅₀ values of 2.57 × 10⁵, 6.03 × 10⁵ and 6.03 × 10⁵, respectively.     

Cultivation of Entomopathogenic Fungi for the Search of Antibacterial Compounds

Entomopathogenic fungi are a rich source of natural bioactive compounds. To establish cultivation conditions which facilitate the production of bioactive compounds and to select good genera among entomopathogenic fungi as the producer, 47 typical entomopathogenic fungi were tested for their ability to produce antibiotic activity. Thirty-eight strains (81%) and 30 strains (64%) of these fungi produced either anti-Bacillus compounds or anti-Staphylococcus compounds, respectively, indicating that the majority of the entomopathogenic fungi tested possessed the ability to produce antibacterial compounds. Using 9 representative strains (Aschersonia sp. HF724, Beauveria bassiana HF338, Cordyceps ramosopulvinata HF746, Metarhizium anisopliae HF293, Metarhizium flavoviride HF698, Nomuraea rileyi HF588, Paecilomyces fumosoroseus HF254, Paecilomyces tenuipes HF419, and Verticillium lecanii HF238), the cultivation conditions in liquid medium were surveyed with respect to the cultivation procedure and medium composition, particularly in terms of the presence or absence of insect-derived materials. At 26 °C, M. anisopliae HF293, N. rileyi HF588, and V. lecanii HF238 strains produced clear antibiotic activity against Bacillus and Saccharomyces, but only in the presence of insect-derived materials, suggesting that the production of antibacterial/antifungal compounds by entomopathogenic fungi is triggered by the presence of insect-derived materials.     

Mitochondrial gene sequences alone or combined with ITS region sequences provide firm molecular criteria for the classification of Lecanicillium species

The recent revision of Verticillium sect. Prostrata led to the introduction of the genus Lecanicillium, which comprises the majority of the entomopathogenic strains. Sixty-five strains previously classified as Verticillium lecanii or Verticillium sp. from different geographical regions and hosts were examined and their phylogenetic relationships were determined using sequences from three mitochondrial (mt) genes [the small rRNA subunit (rns), the NADH dehydrogenase subunits 1 (nad1) and 3 (nad3)] and the ITS region. In general, single gene phylogenetic trees differentiated and placed the strains examined in well-supported (by BS analysis) groups of L. lecanii, L. longisporum, L. muscarium, and L. nodulosum, although in some cases a few uncertainties still remained. nad1 was the most informative single gene in phylogenetic analyses and was also found to contain group I introns with putative open reading frames (ORFs) encoding for GIY–YIG endonucleases. The combined use of mt gene sequences resolved taxonomic uncertainties arisen from ITS analysis and, alone or in combination with ITS sequences, helped in placing uncharacterised Verticillium lecanii and Verticillium sp. firmly into Lecanicillium species. Combined gene data from all the mt genes and all the mt genes and the ITS region together, were very similar. Furthermore, a relaxed correlation with host specificity—at least for Homoptera—was indicated for the rns and the combined mt gene sequences. Thus, the usefulness of mt gene sequences as a convenient molecular tool in phylogenetic studies of entomopathogenic fungi was demonstrated.     

Hydrolytic enzymes secreted by Paecilomyces lilacinus cultured on sclerotia of Aspergillus flavus

Sclerotia, the survival stage of Aspergillus flavus, are compact masses of mycelia capable of with-standing harsh climatic conditions. Six strains of Paecilomyces lilacinus, originally isolated from sclerotia of A. flavus var. flavus or A. flavus var. parasiticus, were also able to colonize the sclerotia from four different strains of A. flavus under laboratory conditions. P. lilacinus strains did not differ significantly in their colonization ability, but host susceptibility appeared to be an important factor. P. lilacinus strains were cultured in vitro for 96 h on a basal salt medium containing either ground sclerotia of A. flavus or glucose plus asparagine. Activities of hydrolytic enzymes such as polysaccharidases, proteases, and chitinases were determined in the culture supernatants. Supernatants from fungal cultures grown in the basal medium containing glucose plus aspargine medium showed very little or no enzyme activity, whereas fungi grown on ground sclerotia produced a variety of enzymes. Specifically, all strains produced chitinases (endochitinase and N-acetyl glucosaminidase), -1,3-glucanase, chymoelastase and chymotrypsin, suggesting that these enzymes may be required for colonization of sclerotia. Production of -1,4-glucanase, dextranase, cellulase, and trypsin was strain variable, suggesting that these enzymes may not be required.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned Correspondence to: S. C. Gupta     

Hyperparasitism of Puccinia horiana and other Microcyclic Rusts

Verticillium lecanii and Aphanocladium album infected in 5 days 90–95 % of the teliospores of Puccinia horiana, the major pathogen of Chrysanthemum. This suggests the possibility to expand the use of V. lecanii, a commercial biocontrol agent against aphids, in order to control P. horiana, V. lecanii and A. album parasitized other microcyclic rusts but with a lesser frequency: P. dianthi (82 and 88 %), P. malvacearum (72 and 60 %) and P. glomerata (57 and 61 %). Cladosporium sphaerospermum and C. uredinicola were less effective than V. lecanii and A. album against all rust species tested.     

Pathogenicity of Three Entomopathogenic Fungi to Matsucoccus matsumurae

Matsucoccus matsumurae (Kuwana) (Hemiptera: Coccoidea: Matsucoccidae) is an invasive alien species and a destructive pest of two native Chinese pines, Pinus tabulaeformis Carr. and P. massoniana Lamb., throughout the eastern regions of China. The pathogenicity of three entomopathogenic fungi, Lecanicillium lecanii strain V3.4504 and V3.4505, Fusarium incarnatum-equiseti strain HEB01 and Lecanicillium fungicola strain HEB02, against M. matsumurae was tested in four instars, to evaluate their potential as a biological control agent. The results showed that the four strains caused disease and death of the scale insect, among which the L. lecanii strains V3.4504 and V3.4505 displayed stronger virulence than the F. incarnatum-equiseti strains HEB01 and L. fungicola strain HEB02 to M. matsumurae in the 2^nd-instar nymphs and the adult females. Furthermore, L. lecanii V3.4505 was most virulent to M. matsumurae. The adult females and the male 3^rd-instar nymphs of M. matsumurae were susceptible to L. lecanii V3.4505; the adult females were more susceptible at LT₅₀ = 1.96 than the 3^rd-instar nymphs at LT₅₀ = 5.67. The body surface structure, cuticle thickness and wax secretions of M. matsumurae impacted the fungal infection. L. lecanii is a promising biocontrol agent, and newly emerged male 3^rd-instar nymphs and adult females are a crucial period of the insect’s life cycle for M. matsumurae biocontrol.     

Development of Transformation System of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: Hyphomycetes) Based on Nitrate Reductase Gene of Aspergillus nidulans

A heterologous transformation system was developed for V. lecanii based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. Mutant no. 01 and 04 was chosen for the transformation experiments. Plasmid pBT was used as transformation vector containing the Aspergillus nidulans nitrate reductase gene. A frequency of approximately 3 transformants/μg DNA was obtained using the circular vector pBT. The establishment of a transformation system for V. lecanii is fundamental for genetic manipulation of this microorganism.     

Glasshouse Experiments on Biocontrol of Cucumber Powdery Mildew (Sphaerotheca fuliginea) by the MycoparasitesVerticillium lecaniiandSporothrix rugulosa

Two potential biological control agents of cucumber powdery mildew (Sphaerotheca fuliginea),Verticillium lecaniiandSporothrix rugulosa,were tested under glasshouse conditions. Two experiments were carried out. In the first experiment, two cucumber varieties with different levels of resistance, cv Corona (susceptible) and cv Flamingo (partially resistant), were used.Verticillium lecaniicontrolled the mildew better thanS. rugulosa.On cv Flamingo,V. lecaniicould keep the mildew severity below 15% infected leaf area for 9 weeks after inoculation withS. fuliginea.Treatment by Hora Oleo 11E, alone or as an additive toV. lecanii,was as good as a fungicide treatment. In the second experiment, weekly and biweekly treatments withV. lecaniiwere compared on cv Flamingo. Weekly treatments withV. lecaniikept mildew severity at a level below 20% infected leaf area during 10 weeks after inoculation withS. fuliginea.If applied to a partially resistant cucumber cultivar,V. lecaniiis an effective candidate for biological control ofS. fuliginea.     

Competitive interaction between strains of Verticillium lecanii on two insect hosts

Differences were observed in the pathogenicities of two strains of Verticillium lecanii (strain numbers 1.72 and 19.79) to the whitefly Trialeurodes uaporariorum and the aphid Macrosiphoniella sanborni. The mean pathogenicity of a mixture of the two strains was intermediate to that of the individual strains, attributable to a competitive interaction during infection. Differences between strains were observed in the production of conidia in vitro and on host insect cadavers. When hosts were infected with the dual-strain suspension, conidia of strain 19.79 only were recovered from T. vaporariorum whereas conidia from both strains were recovered from M. sanborni.     

Formation of aerial hyphae and conidia under orthophosphate-limited conditions inNeurospora crassa: Role of repressible cyclic phosphodiesterase

A wild type strain ofNeurospora crassa produced aerial hyphae and luxuriant conidia in standing culture in low phosphate liquid media.nuc-1 andnuc-2, which have no ability to derepress repressible cyclic phosphodiesterase (cPDase) (3′; 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) and several other repressible enzymes, did not form them. Heterocaryon between them restored the abilities not only to produce aerial hyphae and conidia but also to produce cPDase. Revertants fromnuc-1 and a mutant in alkaline phosphatase,pho-2, produced aerial hyphae and conidia in low phosphate condition, whereas a mutant in cPDase,pho-3, produced only a limited amount of them. In media containing low levels of 2′, 3′-cAMP, the wild type, the revertants fromnuc-1, pho-2 andpho-3 produced aerial hyphae and conidia in abundance, whereas in media containing 3′, 5′-cAMP these strains produced no or only limited amounts of them. In low phosphate medianuc-1, nuc-2 andpho-3 showed higher levels of 3′, 5′-cAMP as compared with those strains which have the ability to derepress cPDase. The cPDase activities in crude mycelial extracts fromnuc-1 andpho-3 grown in low phosphate media were 5.6 and 17.5% of that ofpho-2 when assayed for 3′,5′-cAMP at an intracellular level of 2 μM.     

The ability of soil-borne fungi to degrade organophosphonate carbon-to-phosphorus bonds

The ability of a wide variety of soil-borne fungal strains to degrade four structurally different com pounds containing PC bonds, namely the naturally occurring amino acid ciliatine, the popular herbicide glyphosate, phosphonoacetic acid and 2-amino-3-phosphonopropionic acid, was studied in order to show that soil fungi may play an important role in the biodegradation of organophosphonates. Most of the strains appeared to utilize ciliatine as the sole source of phosphorus for growth. Only a limited number of strains were able to grow on the other phosphonates used in this work. The strains of Trichoderma harzianum, Scopulariopsis sp. and Aspergillus niger chosen for more detailed study show the ability to degrade ciliatine, glyphosate and also amino(3-methoxyphenyl)mehtylphosphonic acid effectively. Received: 14 May 1997 / Received revision: 10 June 1997 / Accepted: 14 June 1997     

Die Parasitierung des Bohnenrostes Uromyces appendiculatus var. appendiculatus durch den Hyperparasiten Verticillium lecanii: Untersuchungen zur Wirt-Erkennung, Penetration und Abbau der Rostpilzsporen

Hyperparasitism of Uromyces appendiculatus var. appendiculatus by Verticillium lecanii Host Recognition, Penetration and Degradation of Spores Culture filtrates of the hyperparasite Verticillium lecanii contain numerous lytic enzymes. When specific substrates were added to the filtrate, degradation of chitin is increased by a factor of 2,25 and degradation of starch is increased by a factor of 1,5. The degradation of uredo- and teliospores of Uromyces appendiculatus var. appendiculatus is documented cytologically. Appressoria-like structures initiated direct penetration of the spore walls. Additional routes of penetration through the germpores of both sporetypes and the pedicles of the teliospores were observed. Degradation of the spore cytoplasm is described. Sugars on the surface of uredo- and teliospores and on hyphae of the hyperparasite were characterized using the gold-marked lectins Con A and WGA. Their role in the host-parasite recognition process is discussed.     

Field Efficacy of Verticillium lecanii,Sex Pheromone,and Pheromone Analogs as Potential Management Agents for Soybean Cyst Nematode

A soybean cyst nematode sex pheromone (vanillic acid), chemical analogs of the pheromone, and the fungus Verticillium lecanii were applied in alginate prills (340 kg/ha) to microplots and small-scale field plots as potential management agents for Heterodera glycines on soybean. In 1991 microplot tests, treatment with V. lecanii, vanillic acid, syringic acid plus V. lecanii, or vanillic acid plus V. lecanii lowered midseason cyst numbers compared with the untreated susceptible cultivar control, autoclaved V. lecanii treatment, or aldicarb treatment, At-harvest cyst numbers were lowest with V. lecanii and with vanillic acid treatments. Aldicarb treatment reduced midseason cyst numbers in 1992. There were no differences among seed yields either year. In the field trials, numbers of cysts were reduced one or both years with aldicarb, ferulic acid, syringic acid, vanillic acid, or 4-hydroxy-3-methoxybenzonitfile treatments, or with a resistant cultivar, compared to an untreated susceptible cultivar. Highest yields were recorded after treatment with 4-hydroxy-3-methoxybenzonitrile (1991), methyl vanillate (1992), and aldicarb (1992). These studies indicate that some chemical analogs of vanillic acid have potential for use in soybean cyst nematode management schemes.     

Activity of pupal parasitoids of the stable fly Stomoxys calcitrans and prevalence of entomopathogenic fungi in the stable fly and the house fly Musca domestica in Denmark

A survey was conducted on confined dairy cattle farms and a pig farm from May–October in 1999 to determine the activity and relative abundance of pupal parasitoids and the prevalence of entomopathogenic fungi in populations of the haematophagous stable fly, Stomoxys calcitrans (Diptera: Muscidae), in Denmark. Four species of pteromalids were found with Spalangia cameroni as the predominant. The other parasitoids were S. nigripes, S. nigra and Phygadeuon fumator (Ichneumonidae). Peak activity of the parasitoids was observed to be late in the summer and the beginning of autumn (August–September) when approximately 10% of the collected stable fly pupae were parasitised. Adult stable flies were infected with four species of entomopathogenic fungi: Entomophthora muscae, E. schizophorae, Beauveria bassiana and Verticillium lecanii. All fungi occurred in low percentages (max. 4%) and remained at this level throughout the sampling period. Likewise, adult house flies were infected with B. bassiana and V. lecanii,but Metarhizium anisopliae, Paecilomyces fumosoroseus and V. fusisporum were also recorded. The overall hyphomycete prevalence in house flies was 0.3%, and single species rarely exceeded 0.1%. The prevalence remained low in spite of increasing house fly numbers in August–September.     

Control of aphids by the fungus, Verticillium lecanii: Effect of spore concentration

In laboratory bioassays, aphid mortality rose while killing time decreased with increasing spore concentrations of Verticillium lecanii. However, in the glasshouse, the ability of the fungus to be spread among aphid populations on chrysanthemums quickly masked the initial effects of spraying different spore concentration. Hundred-fold differences in spore concentrations produced equally good control of the aphid, Myzus persicae, in the glasshouse.

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Controle des pucerons par le champignon: Verticillium lecanii: effet de la concentration en spores

Résumé Dans des essais de laboratoire, la mortalité des pucerons augmente et est plus rapide quand la concentration en spores de Verticillium lecanii s'élève. Cependant, en serre, la possibilité de pulvériser le champignon sur les populations de pucerons sur chrysanthème a masqué rapidement les effets initiaux des différentes concentrations de spores. Ainsi des concentrations de spores différent de cent fois produisent des résultats aussi bons en serre, contre le puceron Myzus persicae.

     

Inheritance of antagonistic properties and lytic enzyme activities in sexual crosses of Talaromyces flavus

Two wild-type strains and three benomyl-resistant mutants of the antagonistic ascomycete Talaromyces flavus were crossed in six combinations, two of which yielded hybrid cleistothecia. Parental strains and their ascospore progenies varied in several traits considered to play an important role in the capacity to control soilborne fungal pathogens: extracellular activities of glucose oxidase and cell-wall degrading enzymes, antibiosis towards Verticillium dahliae, and parasitism and biocontrol of Sclerotium rolfsii. A non-Mendelian quantitative mode of inheritance was found for β-1, 3-glucanase and chitinase activities but only the latter exhibited a normal frequency distribution. Some of the progenies exhibited higher glucanase and chitinase activities than those found in the parental strains. Progeny analysis for chitinase, glucanase, cellulase, and glucose oxidase activities revealed no genetic association between any two of these enzymes. Antibiosis was correlated with glucose-oxidase activity in one cross, but not in the other. The ability to reduce bean root rot caused by S. rolfsii was correlated with mycoparasitic activity against S. rolfsii sclerotia in one cross, but not in the other. One out of the 20 progenies tested was able to reduce bean root rot more effectively than its parent strains, thus demonstrating the feasibility of improving a biocontrol agent by conventional breeding.     

Ostracod reactions to non-toxic and toxic algae

Summary When fed algae continuously, laboratory ostracods had a life span of 21–28 days; without food, they were able to survive about 7 days. When exposed to thick suspensions of gas-vacuolate blue-green algae, survival rates were generally low. Twenty five % of our euplanktonic strains killed entire ostracod populations within 24–48 hrs; about 30% of other waterbloom strains were dangerous to ostracods causing either death or coordination loss. Some amphibious non-gas-vacuolated strains were also toxic to ostracods. Toxins from broken cells were not persistent in effect; ostracods surviving the initial shock often appeared recovered within 2–4 days. In di-algal systems, ostracods generally fed on a single species and did not always choose green algae over blue-green. Unicellular green algal strains (Ankistrodesmus, Scenedesmus, Kirchneriella) were generally preferred. Some chlorophycean forms (Zygnema, Hormidium, Trentepholia) were shunned in favor of bluegreens such as Tolypothrix and Westiella. Erect and branched strains (Fischerella, Westiella, Tolypothrix) were eaten before Anabaena and Nostoc with the latter genus being least preferred. Intact Nostoc colonies were usually not ingested, but some loosely growing, non-toxic strains of both Nostoc and Anabaena provided adequate food for ostracod populations indefinitely.     

Secretion of Extracellular Enzymes by Verticillium psalliotae Treschow and Verticillium lecanii (Zimm.) Viégas During Growth on Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) in Liquid Cultures

Two isolates of the mycoparasite Verticillium psalliotae grew rapidly in liquid cultures on autoclaved uredospores of the soybean rust fungus (Phakopsora pachyrhizi Syd.) as sole carbon source and secreted β-l,3-glucanase, chitinase, and protease activities into the medium. One isolate of Verticillium lecanii grew slowly, failed to produce measurable chitinase activity and secreted lower specific activities of β-l,3-glucanase and protease, compared with V. psalliotae. The tested isolates of V. psalliotae and V. lecanii produced comparable levels of lipolytic activity. Amylolytic activity was secreted by V. lecanii but not by V. psalliotae. The isolates of V. psalliotae and V. lecanii used in our experiments differed clearly in protein and protease pattern, determined by electrophoresis on polyacrylamide gels. The results indicate that the rapid growth of V. psalliotae on autoclaved uredospores in liquid culture and on uredosori is probably based primarily on nutrients made available to the mycoparasite by activities of β-1,3-glucanases, chitinases and proteases.