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.     

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.     

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.     

Verticillium lecanii spore production in solid-state and liquid-state fermentations

Verticillium lecanii has been recognized as an entomopathogen with high potential in biological control of pests. Two types of cultivation methods, the solid-state fermentation (SSF) and the liquid-state fermentation (LSF), were examined for V. lecanii. In SSF, the substrate types including rice, rice bran, rice husk, and the mixtures of these components were tested. The results showed that both cooked rice with appropriate water addition and rice bran gave significantly higher spore production of 1.5 2 10⁹ spores/g substrate and 1.4 2 10⁹ spores/g substrate, respectively. In LSF, SMAY liquid medium was used as a base, and the effects of environmental conditions on the spore production of V. lecanii were investigated. From the time course study, on the 9th day the spore yield reached 1.2 2 10⁹ spores/ml of broth at 24v°C, 150 rpm for this strain. A series of medium volumes in the shaker-flask have been tested for the requirement of aeration. The largest surface aeration test, one tenth of the medium volume in the shaker-flask for cultivation, gave the highest spore count. The optimal pH value was tested and the initial pH 5 in the SMAY medium produced a high spore density. Finally, V. lecanii spores from SSF and LSF were different in size, shape, and size distribution; while mean spore length from SSF was 6.1 7m, and mean spore length from LSF was 5.0 7m.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Optimization of <Emphasis Type="Italic">Verticillium lecanii</Emphasis> spore production in solid-state fermentation on sugarcane bagasse

Verticillium lecanii is an entomopathogen with high potential in biological control of pests. We developed a solid-state fermentation with sugarcane bagasse as carrier absorbing liquid medium to propagate V. lecanii spores. Using statistical experimental design, we optimized the medium composition for spore production. We first used one-factor-at-a-time design to identify corn flour and yeast extract as the best carbon and nitrogen sources for the spore production of V. lecanii. Then, we used two-level fractional factorial design to confirm corn flour, yeast extract, and KH₂PO₄ as important factors significantly affecting V. lecanii spore production. Finally, we optimized these selected variables using a central composite design and response surface method. The optimal medium composition was (grams per liter): corn flour 35.79, yeast 8.69, KH₂PO₄ 1.63, K₂HPO₄ 0.325, and MgSO₄ 0.325. Under optimal conditions, spore production reached 1.1 × 10¹⁰ spores/g dried carrier, much higher than that on wheat bran (1.7 × 10⁹ spores/g initial dry matter).     

Virulence of entomopathogenic Fungi (Fungi imperfecti) for the adults of Acanthoscelides obtectus (Coleoptera: Bruchidae)

Tests with the bean weevil, Acanthoscelides obtectus, in which the hosts were exposed indirectly to various dilutions of conidia of four entomopathogenic fungi showed that mortality was a function of the concentration of the inoculum. In these tests a given spore suspension was sprayed on the internal surfaces of a Petri dish. Adult weevils of a known age were placed in the dish, held there for 24 hr, then removed and kept at 20°C. After 20 days, the host mortality was determined. From the data obtained, it was possible to trace a probit regression line of the mortality in relation to the increasing spore concentration. Infection was observed in hosts exposed to a concentration of approximately 5 × 10⁶ spores/ml up to a maximum of about 1 × 10⁹ spores/ml. The A. obtectus was susceptible to infection by spores of Beauveria bassiana, B. tenella, Metarrhizium anisopliae, and Paecilomyces fumoso-roseus.     

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.     

Factors influencing biocontrol of jimsonweed (Datura stramonium L.) with the leaf-spotting fungus Alternaria crassa

In greenhouse tests, Alternaria crassa (Sacc.) Rands killed > 80% of inoculated jimsonweed (Datura stramonium L.) seedlings within 14 days following a 9-h dew period at 25°C with 1 × 10⁵ spores/ml, and after 8 h of dew at 1 × 10⁶ spores/ml. At least 10 h of dew with 1 × 10⁵ spores/ml and 9 h of dew with 1 × 10⁶ spores/ml were required to obtain 100% mortality of fungus-inoculated plants. Growth stage and inoculum concentration studies revealed that higher concentrations of inoculum were required to obtain 100% mortality of larger plants. Weed control was significantly reduced by day/night air temperatures of 35°C and 24°C, respectively, at all inoculum concentrations as compared to the controls at lower air and dew temperature regimes. The results of these studies indicate that A. crassa has potential as a biological herbicide for the control of jimsonweed.     

The efficacy of the aquatic anaesthetic AQUI‐S® for anaesthesia of a small freshwater fish,Melanotaenia australis

For stage four anaesthesia of the western rainbowfish Melanotaenia australis the most efficacious concentration of AQUI‐S^® was found to be 80 mg l^?1 when five different doses were tested at 27° C (range 26·5–27·5° C). This gave a mean ±s.e. (n = 10) induction time of 140·4 ± 8·9 s and recovery time of 180·2 ± 8·9 s and resulted in 10% mortality when fish were continuously exposed for 15 min. All fish given this dose during induction and recovery time trials ate within 1 h of recovery, and there was zero mortality during a subsequent 1 month monitoring period.     

Sporogenesis of the entomopathogenic fungusVerticillium lecanii in solid-state cultures

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

Influence of <Emphasis Type="Italic">Lecanicillium attenuatum</Emphasis> on the development and reproduction of the cotton aphid, <Emphasis Type="Italic">Aphis gossypii</Emphasis>

The activity of entomopathogens on insect pests has been investigated for many species but the influence of entomopathogenic fungi on factors other than mortality relating to population increase has not been frequently studied. The influence of Lecanicillium attenuatum CS625 (=Verticillium lecanii CS625) on development and reproduction of cotton aphid (Aphis gossypii) was investigated. A conidia suspension of the isolate was applied onto first instar nymphs. Increased spore concentration did not significantly affect each nymphal stage, total nymphal period, pre-reproductive period and the age of first larviposition. A significant dose effect on reduction of life span, reproductive period and fecundity was observed in 1st and 3rd instars after spore application. When conidia were applied to 1st instars, life span was significantly reduced to 10.8 and 8.4 days at 1 × 10⁴ and 1 × 10⁸ conidia/ml, respectively from 12.2 days in the control. During the life span, total fecundity was 41 ± 7.3, 26 ± 0.8 and 22 ± 5.7 nymphs per female at 1 × 10⁴, 1 × 10⁶ and 1 × 10⁸ conidia/ml, respectively compared with 51 ± 2.0 nymphs per untreated female. Reproduction period was also significantly shortened with increasing spore concentration. Application of spores to 3rd instars showed a similar trend. However, daily fecundity of individual aphids was not affected by spore dose. It was concluded that the isolate of L. attenuatum is able to affect populations of cotton aphid by reducing life span and total fecundity as well as by killing the aphids directly.     

Control of glasshouse whitefly, Trialeurodes vaporariorum, by an 'aphid' strain of the fungus Verticillium lecanii

Heavy infestations of whitefly on glasshouse cucumber plants were controlled below the level of economic crop damage by fortnightly or monthly sprays of Verticillium lecanii spores. The fungus did not spread from glasshouse to glasshouse, or from plant to plant, and often not from diseased whitefly scales bearing fungal spore heads to nearby healthy scales. Some scales survived and the resulting adults laid eggs on new leaves bearing no infected scales, creating another, healthy, generation. This makes regular spraying of new leaves essential. Blastospores were as effective as conidia in controlling scales when sprayed to ‘run off’ at concentrations near 10⁷ spores ml^-1 sprayed on to the undersurfaces of leaves. A fivefold increase in spore concentration at levels near 10⁷ spores ml^-1 usually caused significant improvement in mortality, but increase above this concentration is likely to be unrewarding. Thorough coverage of leaves was found to be vital. Control was impaired by dry conditions and by prolonged air temperatures above 25 °C. The fungicide dimethirimol, used against cucumber mildew, did not impair whitefly control by V. lecanii.     

Effects of temperature and dosage on Vairimorpha sp. 696 spore morphometrics,spore yield,and tissue specificity in Heliothis virescens

The effects of temperature and dosage on a new microsporidian species, Vairimorpha sp. 696, were examined in H. virescens. The pathogen was evaluated for tissue specificity, spore size, cumulative percentage mortality, and spore production. All tissues examined bore infection at 32°C. Spore length was significantly longer at 19°C (5.9 μm) than at 32°C (4.7 μm). Spore widths at these two temperatures did not differ significantly. Octospores were not found at either temperature at 8 or 12 days postinoculation. One hundred percent mortality was attained in all dosages administered, but the initial rate of mortaily was more rapid in the higher dosages. Finally, spore yield was greater in larvae administered lower dosages. Maximum spore yield at 27°C was 4.87 × 10⁹ spores/larva.     

Production of microsclerotia of the fungal entomopathogen Lecanicillium lecanii (Hypocreales: Cordycipitaceae) as a biological control agent against soil-dwelling stages of Frankliniella occidentalis (Thysanoptera: Thripidae)

Abstract

Microsclerotia (MS) production by two isolates of Lecanicillium lecanii on various culture media is described, and the efficacy of MS against western flower thrips is evaluated. High concentrations of MS (2.9–3.1×10⁵·mL^?1) were produced in media with C:N ratios of 7.4:1 and 10.3:1 by isolate SN21. Bioassays using soil-incorporated MS resulted in significant infection and mortality of thrips.     

Application ofVerticillium lecanii for the control ofAphis gossypii by a low-volume electrostatic rotary atomiser and a high-volume hydraulic sprayer

Verticillium lecanii (Fungi: Deuteromycete) blastospores were applied to a chrysanthemum crop by an ULV electrostatically charged rotary atomiser (APE-80). The deposition of spores and subsequent control ofAphis gossypii were compared to high volume hydraulic application. A full rate treatment (2×10¹³ blastospores per ha.) was applied by the APE-80 at week 1 and reduced spore rates of 1/6^th and 1/12^th applied by both the APE-80 and the hydraulic sprayer once and twice a week respectively for weeks 1 to 6. Untreated plots served as controls. Initial deposits of spores were higher with the electrostatic sprayer and better distributed with respect to the position of the target aphids. Significantly lower aphid populations were recorded on the electrostatically treated plots in week 4. The single full rate treatment had significantly fewer aphids than the untreated plots from week 3 and all treatments had significantly fewer aphids than the untreated plots from week 5 onwards. The proportion of the aphid population killed byV. lecanii was higher on the electrostatically treated plots until week 6.      

Potential of a granulovirus isolate to control <Emphasis Type="Italic">Phthorimaea operculella</Emphasis> (Lepidoptera: Gelechiidae)

In this study a Brazilian granulovirus strain, PhopGV, isolated from the potato tuber moth (PTM) Phthorimaea operculella, was investigated regarding its potential for biological control and in vivo production. The relationship between mortality of P. operculella larvae and virus concentration was determined at different temperatures on potato tubers and susceptibility of P. operculella to PhopGV was also determined on potato leaves. Virulence of PhopGV to P. operculella was not affected by temperatures from 18 to 30°C. The median lethal concentration (LC₅₀) of larvae fed on potato foliage treated with PhopGV was not higher than that verified with larvae fed on treated tubers. Optimal conditions for production of virus-infected larvae were obtained by using the virus suspensions of 41 × 10⁵, 6.3 × 10⁵ and 62 × 10⁵ OBs ml⁻¹ at 18, 24 and 30°C, which resulted in 32.0, 31.4 and 34.8% of infected larvae collected, respectively. The maximum percentage of infected larvae recovered from tubers was not affected by temperature. However, time for production of virus-infected larvae was longer at 18°C and shorter at 30°C. Persistence of PhopGV was determined on stored tubers and we observed that the virus remained effective for at least two months, causing up to 84.2% mortality of P. operculella at 1 × 10⁷ OBs ml⁻¹. The pathogen was also highly virulent to tomato pinworm, Tuta absoluta, inflicting high percentage of mortality, delaying larval growth and inhibiting pupation. This Brazilian PhopGV strain has potential to control PTM larvae on potato tubers at a broad range of temperature and can be produced in vivo using virus-treated tubers.     

Heliothis zea larval mortality time from topical and per os dosages of Nomuraea rileyi conidia

Heliothis zea larval mortality time was established for topical applications and per os (feeding) with fungal conidia of Nomuraea rileyi. The 6-to 7-day-old larvae received per os dosages ranging from 6 × 10⁶ to 9 × 10⁹ and a topical dosage of ca. 1 × 10⁹. These spore loads initiated fungal infections in larvae resulting in 1.5–100% accumulated mortality during the 30-day testing periods. The data indicated that regardless of the larval treatment method (topical or per os), or conidial dosage rates, the larval mortality time was never shorter than 5 days post-treatment, nor longer than 22 days. The highest percentage of larval deaths occurred from 7 to 9 days with a maximum mortality at 8 days.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.