Fungal extracellular ribotoxins as insecticidal agents

Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen Hirsutella thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and α-sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.     

Exudate from sporulating cultures of Hirsutella thompsonii inhibit oviposition by the two-spotted spider mite Tetranychus urticae

The acaricidal mycopathogen Hirsutella thompsonii has been found to secrete metabolites that are active against femaleTetranychus urticae. Specifically, the rose-colored exudate produced on sporulating cultures of Mexican HtM120I strain sterilized female spider mites in a dose-dependent fashion. Topical application of the exudate resulted in a 100% reduction in mite fecundity over the initial six days of experimentation. Depending upon the exudate dosage, mites partially recovered within 3 and 6 d post-treatment and produced a limited number of eggs. The spider mite active HtM120I exudate contained less detectable HtA toxin than the HtM120I broth filtrate, and it was innocuous when injected into the greater wax moth Galleria mellonella L. larvae. Broth filtrates of HtM120I cultures, although toxic to assayed G. mellonella larvae, did not inhibit mite oviposition to the degree or duration of the exudate preparations. These findings suggest that the factor responsible for suppressing oviposition in female spider mites is linked to the sporulation process and is distinct from the well-characterized HtA produced by vegetative cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Conidiation of Hirsutella thompsonii var. synnematosa in submerged culture

Fourteen monosporal isolates of Hirsutella thompsonii grew vegetatively in various liquid media producing typical phialidic-like conidiophores. Hirsutella thompsonii var. synnematosa from Ivory Coast (HtIC) was the only pathotype which produced true conidia in submerged culture. HtIC began producing conidia after 3 days incubation reaching a peak ranging from 6.8 × 10⁵ to 9.7 × 10⁷ conidia/ml between 6 and 11 days. A concentration of 10 g/liter of corn steep liquor and 0.2% Tween 80 were essential for maximum conidiation. Submerged conidia had a smooth but somewhat rugose conidial walls, whereas aerially formed conidia were distinctly verrucose. Germination of submerged conidia ranged from 5.2 to 12.9% and were virulent causing 32.5% infection to adult citrus rust mites when sprayed on citrus foliage at 1.2 × 10⁹ conidia/ml.     

Isozyme differentiation among 17 geographical isolates of Hirsutella thompsonii

Electrophoretic analysis of mycelial preparations of 17 isolates of Hirsutella thompsonii demonstrated extensive variability in isozyme content. Many isolates possessed distinct electromorphs used to group or separate individual isolates. Coefficients of similarity based on isozyme patterns closely followed the morphological scheme used to separate H. thompsonii into three varieties. One exception, the nonsynnematous vinacious variety was very close to the nonsynnematous grayish-green variety biochemically. The electrophoretic data demonstrate that extensive differentiation among the H. thompsonii isolates is occurring at the subcellular level without attendant morphological changes.     

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.     

The role of native vegetation on infection rates of Calacarus heveae (Acari: Eriophyidae) by Hirsutella thompsonii (Ascomycota: Ophiocordycipitaceae)

Hirsutella thompsonii (Fischer) (Ascomycota: Ophiocordycipitaceae), a fungal pathogen, often causes high mortality in populations of Calacarus heveae Feres (Acari: Eriophyidae), an important pest mite in rubber tree plantations (Hevea brasiliensis Muell. Arg., Euphorbiaceae). However, the ecological and climatic factors regulating this host-pathogen system are poorly known. We compared fungal infections in agroforestry and traditional rubber plantations to evaluate the role of native vegetation and climatic factors on infection rates of C. heveae by H. thompsonii. While the prevalence of H. thompsonii was higher in managed rubber tree plantations, the abundance of C. heveae was about three times higher in traditional plantations. Abundance of C. heveae, agroecosystem management type and microclimatic variables were responsible for driving the infection rates of H. thompsonii. Native vegetation was a source for H. thompsonii and also modified the crop’s microclimate, which contributed to its maintenance in the crop fields. Therefore, appropriate management practices may enhance the effects of entomopathogens on conservative biological control of pest mites in agroforestry systems.     

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.      

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.     

Far-ultraviolet sensitivity and photoreactivation of Hirsutella thompsonii

The far-ultraviolet (FUV; 200–300 nm) sensitivity of four pathotypes of Hirsutella thompsonii, representative of three separate taxonomic varieties of the species, was investigated. The UV inactivation curves for the four strains were indistinguishable. A mutant exhibiting enhanced conidiation derived from the HTF-72 strain was more FUV sensitive than the four wild type strains (fluence enhancement factor ? 1.5). All strains were capable of efficiently photoreactivating FUV damage by a process which appeared to enzymatic.     

Detection of the toxin Hirsutellin A from Hirsutella thompsonii

A total of 162 strains of Hirsutella thompsonii, isolated from infected mites collected worldwide, were examined for the production of Hirsutellin A (HtA). More than half of the broth filtrates exhibited mortality rates superior to 50% when assayed against Galleria mellonella. The presence of the gene coding for HtA, a previously characterized H. thompsonii protein exotoxin, was determined by PCR amplification using gene-specific primers. Most isolates (100 out of 162) were shown to possess the HtA gene. However, the presence of the gene could not be associated with enhanced insecticidal activity. Both isolate groups (with or without an amplifiable HtA gene) produced filtrates that caused the same average mortality rate (65%) when assayed against G. mellonella. The production and secretion of the HtA toxin were estimated by probing broth filtrates with an anti-HtA monoclonal antibody. Again, the detection of the HtA protein was poorly correlated with subsequent mortality rates induced by the broth filtrates of the various H. thompsonii strains. This study suggests that HtA is requisite for neither survival nor pathogenicity, and that H. thompsonii strains are likely to secrete other toxins that have yet to be characterized. Sequencing of a limited number of HtA genes showed that, when present, the gene is highly conserved, and it displays an interesting intronic polymorphism.     

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.     

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.     

The insecticidal protein hirsutellin A from the mite fungal pathogen Hirsutella thompsonii is a ribotoxin

The mite fungal pathogen Hirsutella thompsonii produces a single polypeptide chain, insecticidal protein named hirsutellin A (HtA) that is composed of 130 amino acid residues. This protein has been purified from its natural source and produced as a recombinant protein in Escherichia coli. Spectroscopic analysis has determined that the two protein forms are indistinguishable. HtA specifically inactivates ribosomes and produces the alpha-fragment characteristic of ribotoxin activity on rRNA. Behaving as a cyclizing ribonuclease, HtA specifically cleaves oligonucleotides that mimick the sarcin/ricin loop of the ribosome, as well as selected polynucleotides and dinucleosides. HtA interacts with phospholipid membranes as do other ribotoxins. As a consequence of its ribonuclease activity and its ability to interact with cell membranes, HtA exhibits cytotoxic activity on human tumor cells. On the basis of these results, HtA is considered to be a member of the ribotoxin group of proteins, although it is significantly smaller (130 aa) than all known ribotoxins that are composed of 149/150 amino acids. Ribotoxins are members of a larger family of fungal ribonucleases whose members of smaller size (100/110 aa) are not cytotoxic. Thus, the characterization of the fungal ribotoxin HtA represents an important milestone in the study of the diversity and the function of fungal ribonucleases.     

Genetic diversity in <Emphasis Type="Italic">Hirsutella thompsonii</Emphasis> isolates based on random amplified polymorphic DNA analysis

Hirsutella thompsonii Fisher is a host-specific fungal pathogen of the diverse group of arthropods known as the Acari. It particularly affects eriophyid mites, and the identification of its isolates is a difficult task due to its pleomorphic nature. Seven isolates of H. thompsonii var. thompsonii and H. thompsonii var. synnematosa collected from various agro-climatic regions of India were subjected to PCR analysis using random decamer primers to differentiate between them. Random amplified polymorphic DNA analysis revealed that all of the isolates from the citrus rust mite, Phyllocoptruta oleivora Ashmead (Acari: Eriophyidae), clustered together, with the exception of one, HtCRMB. The only isolate of H. thompsonii from Aceria guerreronis keifer (Acari: Eriophydae), HtPDBC, clustered separately.     

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.     

Toxic effects of spore/crystal ratios of Bacillus thuringiensis on European corn borer larvae

Bioassays to determine LC₅₀ values of spores and crystals of four varieties of Bacillus thuringiensis grown on nutrient agar plates were carried out against neonate and 6-day-old European corn borer, Ostrinia nubilalis, larvae. The four bacterial varieties were equally toxic against the neonates, but only B. thuringiensis var. kenyae, var. galleriae, and var. kurstaki were toxic to 6-day-old larvae. B. thuringiensis var. tolworthi was inactive against 6-day-old larvae. Different ratios of pure spores and crystals of the bacteria also were tested against neonate and 6-day-old larvae. Pure spores are not pathogenic to neonates or 6-day-old larvae. Pure crystals were toxic to both ages of the larvae, but a combination of spores and crystals was necessary for maximum larval mortality.     

A new fungal pathogen of the scavenger mite,Tydeus gloveri

A new fungal pathogen, Hirsutella tydeicola, was found causing epizootics in populations of the scavenger mite, Tydeus gloveri, during the summer of 1979 and 1980 on citrus in Florida. The fungus is described in association with its host using light and scanning electron microscopy. H. tydeicola is compared with a closely related species, H. thompsonii, a coexisting pathogen of the citrus rust mite. All attempts to isolate the fungus on various agar media failed.     

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.     

Isolation of a protein from the parasporal crystal of Bacillus thuringiensis var,kurstaki toxic to the mosquito larva,Aedes taeniorhynchus

The parasporal crystal produced by a strain of Bacillus thuringiensis var. kurstaki (HD-1) contains two serologically distinct proteins. These proteins were isolated from a preparation of the parasporal crystal by Sephacryl S-300 column chromatography. Their molecular weights were estimated as 135,000 and 65,000. Both proteins were toxic to the cabbage looper, Trichoplusia ni, but only the 65,000-dalton protein was toxic to larvae of the mosquito, Aedes taeniorhynchus. Biochemical comparisons based on isoelectric focusing and peptide mapping by two-dimensional gel electrophoresis indicated that the two toxins were distinctly different.     

Purification of Vip3Aa from Bacillus thuringiensis HD-1 and its contribution to toxicity of HD-1 to spruce budworm (Choristoneura fumiferana) and gypsy moth (Lymantria dispar) (Lepidoptera)

We developed a protocol for obtaining high yields (10-15 mg per 1100 ml of culture supernatant) of highly purified (up to 95%) Vip3Aa protein from HD-1 cultures. The protocol is based on acetone precipitation of supernatant protein, followed by HPLC fractionation (DEAE-5PW column) and several concentration steps. Our protocol resulted in higher yields and purity of Vip3Aa than a previously published method [Estruch, J.J., Warren, G.W., Mullins, M.A., Nye, G.J., Craig, J.A., Koziel, M.G., 1996. Vip3A, a 353 novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of 354 activities against lepidopteran insects. Proc. Nat. Acad. Sci. USA 93, 5389-5394.]. This was achieved by using acetone rather than ammonium sulfate for precipitation of proteins from culture supernatants, and a shallow rather than a steep NaCl gradient for elution of the toxin, and by conducting all the purification steps at low temperature to prevent toxin degradation. In bioassays of the purified protein, Choristoneura fumiferana and Lymantria dispar larvae were less susceptible than Spodopteraexigua (10- and ∼100-fold, respectively). A B. thuringiensis var. kurstaki strain HD-1 from which the vip3Aa gene had been deleted (EG12414) showed reduced toxicity to S. exigua relative to the unmodified parental strain (EG2001), but not to L. dispar or C. fumiferana. We interpret these results as indicating that the Vip3Aa toxin does not contribute measurably to pathogenicity of HD-1 in these species.