Susceptibility of Brevipalpus phoenicis to entomopathogenic fungi

The pathogenicity of 52 isolates from several fungus species was studied for the false spider mite Brevipalpus phoenicis. In addition, the main stages during the course of infection by Hirsutella thompsonii, by far the most virulent pathogen, were studied by means of light and electron microscopy. Adult mites were confined to arenas prepared with citrus leaves in acrylic dishes containing agar–water. Conidial suspensions containing 10⁸ conidia/ml were applied, except for H. thompsonii, where a concentration of 10⁷ conidia/ml was used. The H. thompsonii isolates caused higher mortality, with indices higher than 90%. Observations under the scanning electron microscope (SEM) were performed at 0, 6, 12, 24, 48, 72, and 120 h after application of a H. thompsonii suspension containing 10⁷ conidia/ml. Twenty-four hours after inoculation, H. thompsonii conidia were observed attached to the mite’s integument. The conidia germinated and penetrated through the base of the setae on the hysterosoma. Colonization occurred after 48 h, as evidenced by mortality. Conidiogenesis occurred after 120 h, with the development of mycelium and conidiophores emerging from the posterior and anterior parts of the mite.     

Conidioogenous cell differences among mutant and wild-type pathotypes of Hirsutella thompsonii var. thompsonii

Two mutants with different conidiogenesis were isolated from wild phatotypes of Hirsutella thompsonii var. thompsonii collected from the citrus rust mite in Florida. One mutant (WSI) produces more typical single-neck phialides and conidia than the wild type. The other mutant (SS) was developmentally altered in producing condiogenous cells earlier than the wild type as well as phialides with multiple neck capable of conidiogenesis. The mutant (SS) appeared more virulent than mutant (WSI) and the wild type when bioassayed using the citrus rust mite as a host.     

Large-scale production of the fungal pathogenHirsutella thompsonii in submerged culture and its formulation for application in the field

A large-scale method for producing the fungal pathogen,Hirsutella thompsonii, in submerged culture was developed in the laboratory to supply adequate quantities for studies of field efficacy against the citrus rust mite. Laboratory fermentors consisted of 18.9 liter sterilizable solution bottles stopped with aeration heads that were connected in series to supply a closed system for the passage of the sterile compressed air necessary for agitation and for supplying oxygen to the liquid enrichment. The medium contained dextrose at 5 mg/ml, yeast extract and peptone at 5 mg/ml and 0.5 mg/ml, respectively, and essential mineral salts. Dow Corning antifoam V-30 emulsion, and tetracycline hydrochloride (980 mg/g) were included at optimum concentrations of 25 ppm and 5 mg/100 ml, respectively. Each vessel contained 12 liters of media and was inoculated with 75 ml of fragmented mycelia. At 22–26°C, an average of 400 grams (wet wt.) mycelia per vessel were produced after a 96-hr incubation. Upon completion of the fermentation process, mycelia were recovered from the liquid end-products via filtration and stored as mat-mycelia in stainless steel holding pans at 10°C. The mat-mycelia appeared to lose viability some time after 64 days in storage. The sporulation phase of pathogen production was excluded, and the fungus was applied as mycelial fragments by taking the mat from cold storage and formulating it on the day of application. Five hundred gram quantities of mat-mycelia were blended in 500 ml of water for 2 min to form a slurry and then transferred to 18.9 liter solution bottles for transport to the field. All spray formulations (different additives used as spreader stickers and protectants for the mycelia against heat or desiccation) were added and mixed directly in the tank of the sprayer. Formulations containing unsulphured molasses or citrus molasses in combination with Dacagin performed most effectively in field tests. No skin irritation, inhalation difficulties, or symptoms of pathogenicity were experienced by laboratory personnel as a result of exposure toH. thompsonii during the 3-year production effort.     

Beauveria Bassiana Pathogenicity to the Citrus Rust Mite Phyllocoptruta Oleivora

The pathogenicity of Beauveria bassiana to one of the major pests of citrus crops, Phyllocoptruta oleivora, was assessed by inoculating mites with different concentrations of conidia (1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷ and 1×10⁸). Treated mites were kept at controlled conditions (25 ± 0.5°C, 12 h photoperiod and 98% relative humidity) and mite survivorship was evaluated daily. Mortality was found to increase in time and was dependent on the conidia concentration, with values ranging from 24 to 91% for the lowest and highest conidia concentration, respectively. The calculated LC₅₀ on the fifth day was 4.23×10⁶ conidia/ml. Mean lethal time was 3.98, 9.79, 3.09 and 2.74 days for 5×10⁶, 1×10⁷, 5×10⁷ and 1×10⁸ conidia/ml, respectively. Conidia were found to adhere all over the mite body surface, especially at the anal region, where vegetative mycelium was found entering the mite body. We noticed the formation of small crystals inside the mite’s body that were produced during colonization of the body cavity by the fungus. This is the first report of B. bassiana pathogenicity for this species.     

Pathogenesis of infection by the hyphomycetous fungus,Tolypocladium cylindrosporum inAedes sierrensis andCulex tarsalis [Dip.: Culicidae]

The mode of infection and cycle of development ofTolypocladium cylindrosporum Gams was examined inAedes sierrensis andCulex tarsalis. Larvae were found to be infected through the external cuticle, the pharynx and the midgut. Blastospores and conidia were both infective although for equal numerical concentrations blastospores proved more virulent causing high mortality within the first 48 h after inoculation (80 % for L2 larvae exposed to 5×10⁵ spores/ml), while conidia generally took 7–10 days to produce the same results. Sporulation did not occur on submerged cadavers. Conidia were produced only on floating cadavers in contact with air. Conidial production on floating 4th instar larvae was found to average 1.8×10⁷ conidia/larva. Invasion of the haemocoele and fairly extensive growth of the fungus almost invariably occurred before larvae were killed. This was particularly true forAedes sierrensis larvae. Details are presented of growth within the host and post-mortem penetration of the fungus out of the cadaver. AdultA. sierrensis sprayed with a conidial suspension proved susceptible to infection with 100 % mortality being recorded at 10 days. Infections originated in the thorax, suggesting, the integument or possibly the thoracic spiracles to be the most probable site of infection.     

Potential of fungi as biocontrol agents of polyphagotarsonemus latus (Acari: Tarsonemidae)

Infection of broad mites, Polyphagotarsonemus latus, by conidia of Beauveria bassiana, Hirsutella thompsonii, and Paecilomyces fumosoroseus conidia was investigated in the laboratory under controlled temperature and moisture conditions. Infection of P. latus by the fungus (LC₅₀) was related positively to the 1.16 × 10⁶ B. bassiana conidia per ml, 2.39 × 10³ H. thompsonii conidia per ml, and 1.29 × 10⁵ P. fumosoroseus conidia per ml. Mortality caused by B. bassiana occurred the fastest among densities fluctuating between 65 and 125 mites per leaf. The pathogens B. bassiana and P. fumosoroseus, adjuvants (oil and molasses), and the acaricide ABG6364, a microbial insecticide containing the Beta-exotoxin of Bacillus thuringiensis were evaluated for efficacy against P. latus in a greenhouse test. The B. bassiana treated plants had the greatest and most persistent effect on percentage mortality of total broad mites present per leaf (88 %) followed by the acaricide ABG6364. Use of adjuvants (oil and molasses) did not increase infection of P. latus.     

Control of green mould of citrus by using Trichoderma harzianum,humic acid or garlic

Penicillium digitatum, an aggressive fungus causes post-harvest decay of mandarin sweet orange and Washington navel. In vitro Trichoderma harzianum or humic acid (HA) or powdered cloves of garlic caused inhibition of fungal growth of isolates P₁ and P₂. Under storage conditions, the fruit citrus is protected by using T. harzianum with standard volume 2.0?ml (9.6?×?10⁶?conidia/ml) and application 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum spore suspension (1.0?×?10⁶?spores/ml) compared to control. Spraying the fruit citrus by standard volume of 2.0?ml of either HA or powder cloves of garlic 1% on each fruit 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum (1.0?×?10⁶ spores/ml) compared to control. The lowest percentage of disease incidence and disease severity were associated with powder of cloves garlic and followed by HA and T. harzianum during two growing seasons compared with the untreated and control.     

Antifungal effects of citronella oil against Aspergillus niger ATCC 16404

Essential oils are aromatic oily liquids obtained from some aromatic plant materials. Certain essential oils such as citronella oil contain antifungal activity, but the antifungal effect is still unknown. In this study, we explored the antifungal effect of citronella oil with Aspergillus niger ATCC 16404. The antifungal activity of citronella oil on conidia of A. niger was determined by poisoned food technique, broth dilution method, and disc volatility method. Experimental results indicated that the citronella oil has strong antifungal activity: 0.125 (v/v) and 0.25 % (v/v) citronella oil inhibited the growth of 5?×?10⁵ spore/ml conidia separately for 7 and 28 days while 0.5 % (v/v) citronella oil could completely kill the conidia of 5?×?10⁵ spore/ml. Moreover, the fungicidal kinetic curves revealed that more than 90 % conidia (initial concentration is 5?×?10⁵ spore/ml) were killed in all the treatments with 0.125 to 2 % citronella oil after 24 h. Furthermore, with increase of citronella oil concentration and treatment time, the antifungal activity was increased correspondingly. The 0.5 % (v/v) concentration of citronella oil was a threshold to kill the conidia thoroughly. The surviving conidia treated with 0.5 to 2 % citronella oil decreased by an order of magnitude every day, and no fungus survived after 10 days. With light microscope, scanning electron microscope, and transmission electron microscope, we found that citronella oil could lead to irreversible alteration of the hyphae and conidia. Based on our observation, we hypothesized that the citronella oil destroyed the cell wall of the A. niger hyphae, passed through the cell membrane, penetrated into the cytoplasm, and acted on the main organelles. Subsequently, the hyphae was collapsed and squashed due to large cytoplasm loss, and the organelles were severely destroyed. Similarly, citronella oil could lead to the rupture of hard cell wall and then act on the sporoplasm to kill the conidia. Nevertheless, the citronella oil provides a potential of being a safe and environmentally friendly fungicide in the future.     

Effect of entomopathogenic fungi upon adults of Stomoxys calcitrans and Musca domestica (Diptera: Muscidae)

The pathogenicity and virulence of 10 isolates of entomopathogenic fungi from the soil of lodging pens of dairy production units in Aguascalientes, Mexico, on adults of Stomoxys calcitrans and Musca domestica were determined. All isolates were pathogenic when exposed by aspersion to a concentration of 1×10⁸ conidia/ml, causing between 20.3 and 91.7% mortality in S. calcitrans and between 31 and 91.7% in M. domestica at 7 days post-exposure; in S. calcitrans isolates of Beauveria bassiana (Bb114) and Metarhizium anisopliae (Ma135), sensu lato, were the most noteworthy as mortality reached above 90% with an LC₅₀ of 3.5×10⁵ conidia/ml for Bb114, while for Ma135 reached 1.6×10⁴ conidia/ml. In M. domestica Ma134 and Ma135 showed mortality above 90% with an LC₅₀ of 4.3×10⁴ and 1.4×10⁵ conidia/ml, respectively.     

Extraction and Characterization of the Insecticidal Toxin Hirsutellin A Produced by Hirsutella thompsonii var. thompsonii

Liu, W.-Z., Boucias, D. G., and McCoy, C. W. 1995. Extraction and characterization of the insecticidal toxin hirsutellin A produced by Hirsutella thompsonii var. thompsonii. Experimental Mycology 19, 254-262. Hirsutellin A (HtA) produced by Hirsutella thompsonii var. thompsonii (strain JAB-04) was extracted and purified using a combination of ion-exchange, gel-permeation, and immunoaffinity chromatography. The identity of the purified HtA was confirmed by amino acid analysis and N-terminal sequencing. Monoclonal antibodies prepared against HtA were capable of detecting 25-50 ng of HtA by direct sandwich ELISA. In addition, utilizing Western blot methods, the antibodies were shown to be specific to HtA. The production of HtA was monitored during submerged fermentation. The peak level of exocellular HtA (13-14 μg/ml) was during the late exponential growth phase (39-45 h), determined by utilizing a combination of densitometric analysis of the 16.3-kDa bands on SDS-PAGE gels and ELISA. HtA production was directly correlated with mycelial growth. Twenty-one-hour culture filtrates were highly toxic to larvae of the greater wax moth. Pure HtA at a final concentration of 40 pmol was highly toxic to Galleria mellonella larvae.     

Study on the innocuity ofHirsutella thompsonii. I. Infectivity in mice and guinea pigs

The present work concerns the innocuity ofHirsutella thompsonii Fisher isolated in Cuba from a citrus rust mite,Phyllocoptruta oleivora Ashm. This phytopathogenic arachnide is an important pest of different citrus fruit plantations. The infectivity tests were performed in 220 animals (mice and guinea pigs) inoculated by intraperitoneal and subcutaneous injections. Animals showed macroscopic alterations such as nodules in the liver capsule, spleen, subcutaneous tissue; superficial fibrinous adhesions on these organs and hepatosplenomegaly. No fungi were recovered from organs at any time and only disintegrated hyphal elements were observed. The histopathological study disclosed a non-specific tissue reaction to a foreign body. The results obtained suggest the non-infectivity and innocuity ofHirsutella thompsonii Fisher in mice and guinea pigs.      

Lack ofPer os toxicity or pathogenicity in rats fed the fungusHirsutella thompsonii

Symptoms of toxicity or pathogenicity were not observed in white rats fed either whole-culture broth or mycelia of the parasitic fungusHirsutella thompsonii. This fungus is presently under consideration as a microbial control agent of the citrus rust mite,Phyllocoptruta oleivora.     

Hirsutella thompsonii, a fungal pathogen of mites. I. Biology of the fungus in vitro

Hirsutella thompsonii, a moniliaceous fungus pathogenic to mites, grew and sporulated on sterilised wheat bran. The effects of environmental factors were studied on the fungus grown on potato-dextrose-agar (PDA). The fungus was mesothermophilic. Growth, sporulation and conidial germination were best at 25^o-30 ^oC. Conidia kept at 37 ^oC for 5 days on PDA died, but those held at 5 ^oC germinated upon a subsequent removal to 25 ^oC. Almost all conidial germ tubes survived an 8 h exposure to 3–5% r.h. and to 60% r.h., but subsequently the former grew poorly at 100% r.h. H. thompsonii sporulated equally well in continuous darkness or light, and produced typical chlorinous to light olive-green mycelium and conidia under all conditions. A 2 h exposure of naked mycelium and conidia (which have melanised walls) to u.v. irradiation failed to kill the fungus.     

Isolation of entomopathogenic fungi from Northern Thailand and their production in cereal grains

Spore productivity in six entomopathogenic fungal strains isolated from insect cadavers at four locations in Chiang Mai province was evaluated in five cereal grains: white-rice, wheat, rye, corn and sorghum. According to sequence analysis of the internal transcribed spacer regions of these isolates, they were closely related to Beauveria bassiana (2 isolates), Metarhizium flavoviride (1 isolate), Metarhizium anisopliae (1 isolate), Paecilomyces lilacinus (1 isolate) and Isaria tenuipes (1 isolate). Among all fungal isolates, the maximum amount of spores (530.0?×?10⁹ conidia/g) was yielded P. lilacinus CMUCDMT02 on sorghum grain followed by white-rice (399.3?×?10⁹ conidia/g). Moreover, the highest number of spore in M. flavoviride was 102.8?×?10⁹ conidia/g sorghum whereas white-rice yielded the greatest amount of spore for B. bassiana CMUCDMF03 (141.0?×?10⁹ conidia/g) after 60?days incubation. The fungal growth rate was found highest in corn for all strains and rye showed the lowest with the exception of P. lilacinus CMUCDMT02 among the tested grains. Spore viability was over 80?% for all isolates that had been inoculated for 60?days. Fungal conidia suspension of P. lilacinus obtained highest virulence against Bactrocera spp. at a concentration of 1?×?10⁶ spore/ml. The strains isolated, exhibited good production of conidia suggesting a promising strategy for the mass production of inoculum as biocontrol agents with low production cost.     

A Mutant of Metarhizium anisopliae var. acridum with Enhanced Submerged Conidiation

Summary An insertional mutant of Metarhizium anisopliae is described with enhanced submerged conidiation. In a 500 ml submerged culture, this mutant produces a mean of 4.05 × 10⁸ propagules ml⁻¹ from an inoculum of 1 × 10⁶ conidia, where the parental strain accumulates only 3.75 × 10⁴ propagules ml⁻¹.     

Hirsutella thompsonii a fungal parasite of the citrus rust mitePhyllocoptruta oleivora in the Rio Grande Valley of Texas

Hirsutella thompsonii, a hyphomycetous fungus, was found attacking the citrus rust mitePhyllocoptruta oleivora for the first time in May, 1972, in the Lower Rio Grande Valley of Texas. Sharp reductions in mite populations occurred during this period. The fungus was isolated from mites collected from citrus fruits and leaves. The parasitic nature and the distribution of the pathogen are described.     

Host plants influence susceptibility of whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) to the entomopathogenic fungus Isaria fumosorosea (Hypocreales: Cordycipitaceae)

We quantified the tritrophic effect of host plant on the susceptibility of the sweetpotato whitefly Bemisia tabaci (Genn.) to a fungal pathogen in the laboratory. Second-instar whiteflies reared on cucumber, eggplant, tomato and bean plants for six generations were exposed to conidial suspensions of Isaria fumosorosea isolate IF-1106. Our results did not detect differences in response (proportional survival or median lethal time, LT₅₀ days) among insect populations derived from different plants that were treated with 10⁷ conidia/ml. However, at concentrations ≤ 5×10⁶ conidia/ml, whiteflies reared on bean and tomato died significantly more quickly (i.e. LT₅₀ of 4–5 days) compared with cucumber and eggplant reared populations (5–7 days). Bean and tomato-reared populations were also more susceptible to mycosis (LC₅₀ ≈ 6 × 10⁵ conidia/ml) compared with those reared on cucumber (1.9 × 10⁶ conidia/ml) and eggplant (1.5 × 10⁶ conidia/ml). A separate study confirmed that this differential response of whitefly populations to I. fumosorosea was not explained by differences in deposition rate of conidia on leaf surfaces (i.e. a dosage effect). Our findings show that host plants affect the pathogenicity and virulence of a herbivore pathogen, but depend on the rate of exposure (inoculum) applied.     

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.     

Susceptibility ofParacoccidioides brasiliensis conidia to products of oxidative metabolism

The toxic effect of components of the peroxide-peroxidase-halide system onParacoccidioides brasiliensis conidia was investigated. By itself, hydrogen peroxide was lethal at a concentration of 0.5M. The addition of peroxidase (14 U/ml) and KI (5 × 10⁻⁴M) markedly reduced the amount of hydrogen peroxide (from 5 × 10⁻¹ to 5 × 10⁻⁶M) required to kill 99% of the conidia. The lethal effect of the system suggested that it may play a role in host defense againstP. brasiliensis.     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.