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.     

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.     

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.     

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.     

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.

     

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.     

Germination and effect of reduced humidity on expression of pathogenicity in Verticillium lecanii against the glasshouse whitefly Trialeurodes vaporariorum

Fifteen isolates of the entomopathogenic fungus Verticillium lecanii, from various hosts and locations, were bioassayed in the laboratory at 19 ± 1°C and > 95% r.h. against fourth instar scales of Trialeurodes vaporariorum. Highly pathogenic isolates generally originated from whitefly; however, there was no correlation between conidiospore size or germination rate on agar, and pathogenicity. LT₅₀ values ranged from 5.7 to > 9 days and LC₅₀ (7 days) from 1.3 × 10⁵ to 4.2 × 10⁷ spores/ml. The effect of periods in low humidity (70% r.h.) following 16 or 96 h at > 95% r.h. after immersion of whitefly scales on tomato leaf discs in a suspension of 1 × 10⁶ spores/ml, was compared for five isolates. Progressively longer periods at 70% r.h. following 16 h at > 95% r.h., reduced significantly (P < 0.001) the pathogenicity of all isolates. In these conditions isolate A was least affected by low-humidity transfer. The high pathogenicity of isolate A was associated with its more rapid development on the host cuticle during the first 16 h in high humidity. When inoculated whitefly scales were transferred to 70% r.h. after a preliminary 96 h incubation in high humidity, all five isolates achieved a higher level of pathogenicity compared with their transfer to low humidity after 16 h high humidity. Thus the screening of V. lecanii isolates in limiting humidity conditions provided a more critical assessment of their pathogenicity and of their potential success in whitefly control relevant to the glasshouse environment.     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

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.     

Strobilurin + acibenzolar-S-methyl controls white rust without inducing leaf chlorosis in spinach

White rust (Albugo occidentalis) is a major foliar fungal disease of spinach (Spinacea oleracea) in Texas and the Southeastern USA. It causes leaf yellowing, decreasing product quality and marketability. There is a need for evaluating broad spectrum fungicides and new plant defence activators that have the potential to enhance leaf quality in spinach. Two of those fungicides are acibenzolar‐S‐methyl (ACIB) and strobilurin derivatives (STRO). ACIB appears to mimic natural systemic acquired resistance response in plants, while STRO inhibits mitochondrial respiration in fungi. We conducted three consecutive studies to a) determine the effects of ACIB rates and combinations with STRO and mefenoxam + copper hydroxide (MEFE‐Cu) for the control of white rust, and b) evaluate potential phytotoxicity expressed as leaf chlorosis. After two weekly applications in 1998, ACIB, STRO and ACIB + STRO significantly reduced the % of leaf area infected with white rust in the spinach cv. Cascade. In 1999, ACIB + STRO and ACIB + MEFE‐Cu had the best white rust efficacy, with ACIB equally effective when applied at reduced rates. After three applications in 2000, STRO, ACIB + STRO, and ACIB + MEFE‐Cu had significantly lower white rust values than the control, ACIB or MEFE‐Cu. The combination of ACIB + STRO did not cause leaf chlorosis in any year. Use of preventive applications of ACIB + STRO at 7–10 day intervals appear to be an effective strategy to improve leaf quality in spinach.     

White rust of chrysanthemums

Teleutospores of Puccinia horiana Henn. germinate and discharge sporidia between 4 and 23 °C. At the optimum temperature of 17 °C sporidia discharge starts within 3 h. Maximum germination of the sporidia takes place within 2·5 h between o and 30 °C, there being no clear optimum. High humidity and a film of moisture appear to be necessary for germination of both teleutospores and sporidia. Sporidia can penetrate either leaf surface of chrysanthemum to cause infection between 4 and 24 1°C and within the optimum temperature range, 17–24 °C, effectively penetrate within 2 h. The sporidia are very sensitive to desiccation at below 90 % relative humidity. Methods are described, using leaf discs and whole plants, for screening chrysanthemum cultivars for susceptibility to white rust. Cultivars were placed in five classes ranging from susceptible to immune. Leaf discs of immune cultivars can be distinguished within 30 h by a brown discolouration at the point of inoculation. The early stages of development of the fungus in susceptible, resistant and immune hosts are described. The incubation period in susceptible plants is normally 7–10 days, teleutospores being formed a few days later. Leaves become less susceptible with age but the oldest leaves on 5-month-old plants could still be infected. The maximum survival time of teleutospores in the sori on detached leaves was 8 weeks but was considerably less under moist conditions or buried in soil. Low doses of a mancozeb with zineb fungicide controlled infection by preventing penetration rather than by inhibiting sporidial germination.     

Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum × morifolium based on rDNA ITS sequence analysis

Isolates of the most important Puccinia species that have been reported on Chrysanthemum × morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.     

Development and validation of CAPS and AFLP markers for white rust resistance gene in<Emphasis Type="Italic"> Brassica juncea</Emphasis>

White rust, caused by Albugo candida, is a very serious disease in crucifers. In Indian mustard (Brassica juncea), it can cause a yield loss to the extent of 89.9%. The locus Ac2(t) controlling resistance to white rust in BEC-144, an exotic accession of mustard, was mapped using RAPD markers. In the present study, we developed: (1) a more tightly linked marker for the white rust resistance gene, using AFLP in conjunction with bulk segregant analysis, and (2) a PCR-based cleaved amplified polymorphic sequence (CAPS) marker for the closely linked RAPD marker, OPB06₁₀₀₀. The data obtained on 94 RILs revealed that the CAPS marker for OPB06₁₀₀₀ and the AFLP marker E-ACC/M-CAA₃₅₀ flank the Ac2(t) gene at 3.8 cM and 6.7 cM, respectively. Validation of the CAPS marker in two different F₂ populations of crosses Varuna × BEC-144 and Varuna × BEC-286 was also undertaken, which established its utility in marker-assisted selection (MAS) for white rust resistance. The use of both flanking markers in MAS would allow only 0.25% misclassification and thus provide greater efficiency to selection.Communicated by C. Möllers     

Molecular markers linked to white rust resistance in mustard Brassica juncea

 White rust, caused by Albugo candida (Pers.) Kuntze, is an economically important disease of Brassica juncea (L.) Czern. and Coss mustard, particularly in India. The most efficient and cost-effective way of protecting mustard plants from white rust disease is through genetic resistance. The objective of this study was to identify RAPD markers for white rust resistance in an F₁-derived doubled-haploid (DH) population originating from a cross between white rust-susceptible and white rust-resistant breeding lines of B. juncea from the canola-quality B. juncea breeding project of the Agriculture and Agri-Food Canada-Saskatoon Research Centre. The DH population was used to screen for RAPD markers associated with white rust resistance/susceptibility using bulked segregant analysis. Two markers, WR2 and WR3, linked to white rust resistance, flanked the resistance locus Ac2 ₁ and were highly effective in identifying the presence or absence of the resistance gene in the DH population. These two markers were shown to be specific to the Russian source of white rust resistance utilized in this project. It is concluded that the availability of these RAPD markers will enhance the breeding for white rust resistance in B. juncea. Received: 17 December 1997 / Accepted: 7 April 1998     

Insecticidal activity of Poppy (Papaver somniferum L.) seed oil against cowpea weevil (Callosobruchus maculatus F.) in stored cowpea

In this study, the insecticidal effect of Poppy (Papaver somniferum) seed oil was investigated against Callosobruchus maculatus (Bruchidae) in cowpea, at different concentrations and exposure time. The Poppy seed oil at 2, 4, 6, 8 and 10?ml/kg was tested against adults of C. maculatus, and the mortality was counted after 24, 48 and 72?h of exposure. All tests were conducted at 27–30?°C and 65?±?5% r.h. The experiments were carried out based on the factorial experiment by randomized complete design with four replications. Twenty-five insects with 0–24?h old were used for each replication. The effect of Poppy seed oil on the reduction of emergence of insects in next generation (F₁) was also assayed. The results of experiments indicate the significant differences between concentrations and exposure time (p?<?0.0001), and the increase of concentrations and exposure time increased mortality. The amount of mortality at high concentrations was remarkable, and highest mortality rate (96.91%) was recorded at 10?ml/kg, after 72?h of exposure. The application of oil significantly reduced F₁ progeny production and even at lowest concentration (2?ml/kg), the amount of F₁ production was reduced more than 70%, compared with control treatment. Complete (100%) reduction in progeny production was recorded at the rates of 8 and 10?ml/kg. The results of seed germination assay showed no significant differences between control and treated seed, and no harmful effect was observed on the seed germination. These results proved that Poppy seed oil can be used as a controlling agent of storage pests, especially C. maculatus, although more detailed studies are necessary.     

Laboratory studies on the fungus Verticillium lecanii, a larval pathogen of the large elm bark beetle (Scolytus scolytus)

From 1972 to 1974, estimates of the natural larval mortality (> second instar) of elm bark beetles caused by pathogenic organisms were always below 7'5 % of the beetle population. The pathogenic fungus Verticillium lecanii was frequently isolated from field-collected dead larvae, and in the laboratory all larvae were killed in 5 days when exposed to spore concentrations of 4·5 × 10⁶ spores/ml. V. lecanii begins to lose its pathogenicity after prolonged culture on artificial media. The time taken for V. lecanii to kill Scolytus scolytus larvae when exposed to a logarithmic series of spore dilutions from 9·1 × 10⁷/ml to 9·1 × 10³/ml increased with decreasing amounts of inoculum. Even at spore concentrations as low as 9·1 × 10³/ml the mortality of treated larvae was greater than that of untreated individuals. At 100% r.h. all treated larvae were killed over a temperature range of 5–30 °C; those maintained at 25 °C were killed most rapidly and those kept at 5 °C the slowest.     

Susceptibility of the tobacco whitefly,Bemisia tabaci (Hemiptera: Aleyrodidae) biotype Q to entomopathogenic fungi

Recently, the Q biotype of tobacco whitefly has been recognized as the most hazardous strain of Bemisia tabaci worldwide because of increased resistance to some insecticide groups requiring alternative strategies for its control. We studied the susceptibility of this biotype of B. tabaci to 21 isolates of Beauveria bassiana, three isolates of Isaria fumosorosea, one isolate of I. cateni, three isolates of Lecanicillium lecanii, one isolate of L. attenuatum, and one isolate of Aschersonia aleyrodis. These isolates were evaluated on pruned eggplant seedlings, at a concentration of 10⁸ conidia/mL (deposited at 6000±586 conidia mm^?2). The mortality based on mycosis varied from 18 to 97% after 6 days. Isaria fumosorosea isolate Pf04, B. bassiana isolates Bb06, Bb12, and L. lecanii L14 were found the most effective. Furthermore, five isolates were chosen for concentration–mortality response assays and compared to B. bassiana GHA as a standard. The numbers of nymphs infected by fungi were correlated with the spore concentration. L. lecanii L14 and I. fumosorosea Pf04 had the shortest LT₅₀ at 3.5 and 3.3 days at 6000±586 conidia mm^?2. Mortality declined and LT₅₀ values were longer as the concentration of conidia was reduced. The LD₅₀ values were calculated as 87, 147, 191, 263, and 269 conidia mm^?2 for isolates L14, GHA, AS1260, Bb13, and Pf04, respectively. These results indicated that the Q biotype of sweetpotato whitefly was susceptible to the five isolates of entomopathogenic fungi and these isolates have potential to be developed as microbial pesticides for whitefly control.     

Relationship between agitation speed and the morphological characteristics of Verticillium lecanii CS-625 during spore production

Verticillium lecanii is recognized as an entomopathogenic fungus, and has high potential in the biological control of pests. In this study, it was investigated that the relationship between agitation speed in a 2.5 L stirred tank reactor (STR) at 25°C and initial pH 5.5, and the morphological characteristics of V. lecanii CS-625, such as hyphal length/width, spore length/width, and the number of tips during spore production. The agitation speed affected the hyphae patterns and the number of tips. The number of spores rapidly increased at 48 to 60 h of cultivation, and the highest spore productivity (2.5 × 10¹⁰ spore/L·h at 60 h) occurred with an agitation speed of 350 rpm and an aeration rate of 1.0 vvm. The number of tips increased in proportion to the increase in spore production during the same culture time. The highest number of tips (4.8 × 10⁸ tipJ.mL) was obtained at 72 h of cultivation. The shortest mean spore length (2.8 μm) was obtained at 60 h of cultivation. Therefore, it was determined that the increased number of tips and decreased mean spore length were closely related to the production of V. lecanii spores.     

New records of entomopathogenic fungi infecting Bemisia tabaci and Trialeurodes vaporariorum, pests of horticultural crops, in Argentina

The whiteflies Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) are major crop pests throughout the world. Although extensive research about biological control of whitefly by parasitoids and predators has been conducted, also entomopathogenic fungi can be considered as potential biological control agents. Surveys for entomopathogenic fungi were carried out in organic and conventional horticultural crops in greenhouses and open fields in Buenos Aires and Corrientes provinces, Argentina. These surveys resulted in the recovery and isolation of the following fungi from whiteflies: Lecanicillium lecanii (Zimmerm.) Zare & W. Gams, L. muscarium (Petch) Zare & W. Gams, L. longisporum (Petch) Zare & W. Gams, Isaria fumosorosea Wize and I. javanica (Frieder. & Bally) Samson & Hywel-Jones. Pathogenicity tests were conducted against T. vaporariorum nymphs using a conidial suspension (1 × 10⁷ conidia/ml) of the fungi. A mortality rate between 26.6% and 76.6% was obtained at 7 days post-infection. These are the first records of natural infections in the southernmost region of the South American continent of L. lecanii, L. muscarium, L. longisporum and Isaria javanica (Ascomycota: Hypocreales) on T. vaporariorum and also the first report of I. fumosorosea on B. tabaci.