Degradation of Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) by Cell-Free Culture Filtrates of the Mycoparasite Verticillium Psalliotae Treschow

Verticillium psalliotae isolates Taiw and Thai C are effective parasites of the soybean rust fungus. Cell-free culture filtrates of these fungi, prepared after growth on autoclaved uredospores, contained β-1,3-glucanase, chitinase and protease activities and caused degradations, when rust spores were treated with them for 24 or 72 h. During these lytic processes carbohydrates, amino compounds and N-acetylhexosamine were released. The carbohydrate fraction was composed of mannitol, arabitol, trehalose, glucose and unidentified substances showing low R_f-values during thin layer chromatography. The amino compounds consisted of 10 amino acids (leucine and/or isoleucine, phenylalanine, glutamic acid, valine, arginine, asparagine, glutamine, serine, aspartic acid, histidine) and 5—7 substances which could not be identified. Verticillium lecanii isolate Konz is a weak parasite of soybean rust. During growth on uredospores the fungus produced culture filtrates without chitinase activity and with low total activities of β-1,3-glucanase and protease. Compared with V. psalliotae, culture filtrates of V. lecanii exerted lower lytic activities on soybean rust uredospores. The results are consistent with the aspect that the rapid growth of V. psalliotae on soybean rust fungus is primarily based on the secretion of lytic enzymes which make nutrients available to the mycoparasite.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

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.     

Traits associated with virulence to the aphid Macrosiphoniella sanborni in eighteen isolates of Verticillium lecanii

Eighteen isolates of the entomopathogenic fungus Verticillium lecanii from various world-wide locations, from insect hosts and soil were bioassayed against the aphid Macrosiphoniella sanborni in the laboratory. Virulence ranged from an isolate which achieved 100% mortality and LT 50 value (adults) of 3 days ± 0· 2 at 24 ° C, compared with the least virulent isolates causing less than 10% mortality over 14 days (when treated with an inoculum of 1 × 10⁶ conidiospores/ml). All isolates produced extracellular protease and lipase, irrespective of their virulence. A number of traits were frequently associated with the expression of virulence including fast germination, high sporulation rate, an absence of extracellular amylase activity and high extracellular chitinase activities. Large spore size was not strongly associated with virulence. There were exceptions in each variate studied, suggesting that overall expression of virulence is a result of the total complex of these and other traits still to be determined.     

Chitinase Activity in Stylosanthes guianensis Systemically Protected Against Colletotricbum gloeosporioides

Three- and four-fold increases in chitinase activity were detected in the youngest, fully expanded leaf (L₁) of Stylosanthes guianensis cv. Endeavour following inoculation of the second youngest, fully expanded leaf (L₂) with virulent Type B and Type A isolates of Colletotrichum gloeosporioides compared with chitinase activity in L₁ leaves of uninoculated plants. Only small increases in β-1,3-glucanase were detected in L₁ leaves of systemically protected plants. Chitinase activity was maximal in leaf L₁ 36 to 48 h after inoculation of leaf L₂, and this was coincident with the onset of resistance to anthracnose in L₁ leaves. Chitinase activity also increased in L₁ leaves inoculated with a weakly pathogenic isolate of C. gloeosporioides. Resistance developed in these L₁ leaves to subsequent infection by a virulent isolate of the pathogen approximately 36 h after protective-inoculation with the weakly pathogenic isolate. Two chitinase isozymes, with molecular weights of 65,000 daltons (pI 3.1) and 54,000 daltons (pI 4.0), were separated from extracts of C. gloeosporioides-challenged S. guianensis cv. Endeavour leaves. S. guianensis chitinase caused death of C. gloeosporioides hyphae, particularly in the presence of β-1,3-glucanase. Mycelial viability declined as activity of chitinase was increased in mixtures containing a fixed activity of β-l,3-glucanase.     

Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

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.     

Characterization of an Extracellular Serine Protease Gene from the Nematophagous Fungus <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis>

The gene encoding a cuticle-degrading serine protease was cloned from three isolates of Lecanicillium psalliotae (syn. Verticillium psalliotae) by 3′ and 5′ RACE (rapid amplification of cDNA ends) method. The gene encodes for 382 amino acids and the protein shares conserved motifs with subtilisin N and peptidase S8. Comparison of translated cDNA sequences of three isolates revealed one amino acid polymorphism at position 230. The deduced protease sequence shared high degree of similarities to other cuticle-degrading proteases from other nematophagous fungi.     

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 Mg²⁺ and Ca²⁺ concentration. The protease showed inhibitory activity against several plant pathogens, especially towards Fusarium moniliforme.     

A mechanistic investigation of the microbial chiral inversion of 2-phenylpropionic acid by Verticillium lecanii

Previous investigations have described the development of nongrowing suspension of Verticillium lecanii as a microbial model of the mammalian chiral inversion of the 2-arylpropionic acids (2-APAs). Mechanistic studies in mammals have shown that inversion involves loss of the α-methine proton but retention of the original atoms at the β-methyl position, and a mechanism has been proposed involving enzymatic epimerisation of acyl-CoA thioester derivatives of the substrate. Inversion of the 2-APAs by V. lecanii exhibits extensive intersubstrate variation in the presence, rate, extent, and direction of inversion, which are different from those observed in mammalian systems, possibly indicating differences in the mechanism of inversion between mammalian and microbial cells. This study involved the investigation of proton/deuterium exchange by ¹H-nuclear magnetic resonance following incubation of deuterated derivatives of 2-phenylpropionic acid (2-PPA), a model compound, in cell suspensions of V. lecanii and incubation of undeuterated 2-PPA in cell suspensions containing D₂O. The results indicated that the inversion of 2-PPA by V. lecanii also involved exchange of the α-methine proton but complete retention on the original atoms at the β-methyl position. No kinetic deuterium isotope effect was observed, indicating that loss of the α-methine proton is not the rate-limiting step of the inversion process. This suggests that the observed differences between microbial and mammalian systems probably involve the stereoselective acyl-CoA thioester formation step and not the subsequent epimerisation of the resultant diastereomers. Chirality 9:254–260, 1997. © 1997 Wiley-Liss, Inc.     

Einsatz von V. lecanii als biologisches Schädlingsbekämpfungsmittel gegen den Bohnenrostpilz U. appendiculatus var. appendiculatus im Feld und im Gewächshaus

The use of Verticillium lecanii as a biological control agent against the bean rust fungus Uromyces appendiculatus var. appendiculatus in the field and in the glasshouse The deuteromycete V. lecanii parasites uredo- and teliospores of the bean-rust-fungus U. appendiculatus var. appendiculatus. We investigated the conditions for the use of the hyperparasite as biological control agent in the field and in glasshouses. The growth rate of the hyperparasite was 0,3 cm per day at 25 °C. Under suitable conditions in the lab (25 °C, 100 % r. h.) it took about 20 days to invade 100 % of uredospores and 65 % of teliospores. We failed to prevent the spread of bean-rust-fungus spores in the field, but we succeeded in the glasshouse by 68 %, compared to the untreated controls, using the hyperparasite V. lecanii as biological control agent.     

Molecular Characterization of the Host-Adapted Pathogen Verticillium longisporum on the Basis of a Group-I Intron Found in the Nuclear SSU-rRNA Gene

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and non-phytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies. Received: 24 August 2000 / Accepted: 25 September 2000     

A review of white rust (Puccinia horiana Henn.) disease on chrysanthemum and the potential for its biological control with Verticillium lecanii (Zimm.) Viégas

The biology, aetiology and epidemiology of Puccinia horiana, the cause of white rust disease of chrysanthemum (Chrysanthemum spp.) is reviewed in relation to current environmental, cultural and chemical methods for its control. Importantly, basidiospore release, germination and infection can take as little as 5 h at optimum r.h. (96%) and temperature (between 17–24°C). Recent developments using the fungus Verticillium lecanii for the control of insects on glasshouse-grown all-year-round chrysanthemums rely upon the maintenance of r.h. during night periods in excess of 95%, thus predisposing plants to white rust attack. However, V. lecanii is unusual in that it can also parasitise spores and fruiting structures of a range of rust fungi including P. horiana. This mycoparasitic ability is also reviewed, and against this background, the potential for an integrated insect and white rust control programme on all-year-round chrysanthemums is assessed.     

A screening strategy of fungal biocontrol agents towards Verticillium wilt of cotton

Three hundred and seventy-three fungal isolates were obtained from the endorhiza, rhizosphere, and bulk soil of field-grown cotton plants. One hundred and five of them produced obvious inhibition zones against Verticillium dahliae Kleb., so they were selected as antagonists towards this pathogen. An assessment system was established to evaluate these 105 antagonists for their biocontrol potential and plant growth-promoting potential. Their biocontrol potential was assessed according to their in vitro antagonistic activity against V. dahliae and activities of fungal cell wall degrading enzymes including protease, cellulase, and chitinase. Their plant growth-promoting potential was assessed according to their in vitro activities of solubilizing phosphate and fixing nitrogen. Thirty-three antagonists received at least three points of the total value of assessed biocontrol potential and plant growth-promoting potential and were tested for their biocontrol efficacy and growth-promoting effect on cotton under greenhouse conditions. Twelve of them achieved positive biocontrol efficacy ranging from 8.58% to 69.78%; the conventional correlation coefficient of the biocontrol efficacy of these antagonists with their assessed biocontrol potential was 0.926. By using the screening strategy developed in this study, Fusarium oxysporum strain By125, Nectria haematococca Bx247, and Phomopsis sp. By231 were identified as potential BCAs for controlling Verticillium wilt in cotton, for they achieved biocontrol efficacy of 63.63–69.78% towards this disease and increased biomass by 18.54–62.63% under greenhouse conditions. The present study also demonstrated that the endorhiza of field-grown cotton plants may be a richer source of potential BCAs against Verticillium wilt than the rhizosphere and bulk soil.     

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.     

Application of a Sex Pheromone,Pheromone Analogs,and Verticillium lecanii for Management of Heterodera glycines

A mutant strain of the fungus Verticillium lecanii and selected bioregulators of Heterodera glycines were evaluated for their potential to reduce population densities of the nematode on soybean under greenhouse conditions. The bioregulators tested were the H. glycines sex pheromone vanillic acid and the pheromone analogs syringic acid, isovanillic acid, ferulic acid, 4-hydroxy-3-methoxybenzonitrile, and methyl vanillate. A V. lecanii-vanillic acid combination and a V. lecanii-syringic acid combination were also applied as treatments. Syringic acid, 4-hydroxy-3-methoxybenzonitrile, V. lecanii, V. lecanii-vanillic acid, and V. lecanii-syringic acid significantly reduced nematode population densities in the greenhouse tests. Results with vanillic acid, isovanillic acid, and ferulic acid treatments were variable. Methyl vanillate did not significantly reduce cyst nematode population densities in the greenhouse tests.     

Activation of Glycosidases as a Consequence of Infection Stress in Fusarium Wilt of Tomato

Changes of β-1,3-glucanase, chitinase, β-1,4-glucosidase and N-acetylglucosaminidase activity have been investigated in relation to the development of symptoms and colonization by the pathogen in roots, stems and leaves of susceptible (‘Improved, Pearson’) and resistant (‘Improved Pearson VF11’) tomato plants infected by Fusarium oxysporum f. sp. lycopersici. Glycosidase activities increased after inoculation to different extents depending on the plant part and cultivar. Increases were always higher in susceptible than in resistant plants. Changes in the β-1,3-glucanase activity after inoculation were particularly large in stems of infected plants. In contrast, chitinase activity increased more in roots than in stems. The β-1,3-glucosidase and chitinase activity decreased slightly from the basal to the apical third of stems. The trend of changes of the glycosidase activity generally were well related with the severity of disease symptoms and the fungal colonization of basal stem segments. There was no evidence that the increase of glycosidase activity after the infection was directly related with the resistance to Fusarium wilt in tomato.     

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.