Formation and germination of Entomophaga maimaiga azygospores

Azygospores (resting spores) of the gypsy moth fungal pathogen Entomophaga maimaiga are produced in abundance during late spring and early summer in late-instar gypsy moth larvae (Lymantria dispar). Azygospores subsequently form, each from an individual hyphal body. Development of azygospores occurs asynchronously over several days; by 5 days after host death, greater than 60% of fungal cells had matured from hyphal bodies to the final double-walled resting state. Azygospores undergo constitutive dormancy and, under field conditions, will not germinate for approximately 9 months after production. Azygospores do not require nutrients to germinate. Germination of field-collected resting spores under laboratory conditions began more than 2 days after transfer from the field to the laboratory. Higher levels of germination occurred with a 14 h L: 10 h D cycle compared with 13 h L: 11 h D or 12 h L: 12 h D. Azygospores germinate relatively slowly and germination rates were greatest between 4 and 8 days, with a total of 71.8 or 72.5% germination by 16 days at 14 h L: 10 h D and 15 or 20 °C, respectively. During 1994 and 1995, resting spores began causing infections in experimental larvae in early May, about 1 – 2 weeks prior to gypsy moth egg hatch, and ceased causing infections in mid to late June, when late instars were present. This latter timing is a correction of previously reported information. Bioassays investigating resting spore activity determined that during 1994, once resting spores began germinating in the field, levels of infection were positively associated with soil moisture. Key words: azygospores, resting spores, entomopathogenic fungi, Entomophaga maimaiga, Lymantria dispar, biological control.

Physiological studies with the fungus Entomophaga aulicae during morphogenesis in three different media under fermentation conditions

The effects of three different media on amino acid uptake and production and glucose and oxygen utilization during protoplast growth and hyphal body production by the fungus Entomophaga aulicae under fermentation conditions were studied. The three media consisted of a basal medium plus either (i) 2.8% fetal calf serum, (ii) 0.8% tryptic soy broth plus 0.4% bovine serum albumin, or (iii) 0.8% tryptic soy broth plus 0.4% calcium caseinate. The protoplasts grew most rapidly (initial peaks on days 2 and 3) and hyphal bodies were detected first (day 3) in the media containing albumin and caseinate. The day 9 hyphal body yields were 3.1 × 107, 7.5 × 108, and 3.1 × 109/10 L in media containing the serum, albumin, and caseinate, respectively. Growth in the albumin and caseinate media also gave the first detectable glucose utilization (days 2 and 3, respectively) and this rapidly increased to 94.9 and 90.6% utilization, respectively, on day 4. Oxygen and glucose utilization were closely related. During protoplast growth prior to hyphal body production, the only common pattern detected was the initial utilization of glutamine in serum- and caseinate-containing media. During the initial period of hyphal body production, cysteic acid, threonine, serine, asparagine, leucine, tyrosine, phenylalanine, and arginine were first utilized and glycine, alanine, and ammonia were first produced in the albumin and caseinate media. At this time (days 3–5), glutamine, proline, cystine, and tryptophan were first utilized and valine and histidine were produced in the albumin medium, and methionine was first utilized and cystathionine produced in the caseinate medium. Four main patterns of overall amino acid utilization and production were identified. The delay in major protoplast growth in the basal medium plus fetal calf serum is felt to result from inhibition by free fatty acids in the serum. Protein utilization was not detected and its main function is considered to be enhancement of protoplast stability against fermentation shear forces.Key words: Entomophaga aulicae, physiology, fermentation growth, protoplasts, hyphal bodies.

An inexpensive medium for mass fermentation production of Entomophaga aulicae hyphal bodies competent to form conidia

A mass fermentation medium for growth and morphogenesis of the entomopathogenic fungus Entomophaga aulicae was developed. This fungus is a major pathogen of larval eastern hemlock looper and spruce budworm. The medium consists of a basal medium plus 0.8% tryptic soy broth and 0.4% calcium caseinate. This medium is a major breakthrough in that (i) the E. aulicae developmental sequence from protoplast inoculum to hyphal bodies competent to form conidia can be carried out in a single medium without adjustment, (ii) by examining the fermentation product it can be determined if conidia can be produced prior to engaging in costly field spraying, (iii) this medium supports the growth of E. aulicae isolates from different geographical areas, (iv) the medium is relatively inexpensive, (v) the hyphal bodies are easily separated from the spent growth medium, and (vi) the hyphal body yield is high.Key words: Entomophaga aulicae, mass fermentation medium, hyphal bodies, conidia, insect biocontrol.

Ultrastructural localization of carbohydrate in cell walls of the entomogenous hyphomycete Nomuraea rileyi

Several probes were used in this ultrastructural study to localize polysaccharides in cell walls on conidial germ tubes, hyphal bodies, and mycelia of the entomogenous hyphomycete Nomuraea rileyi. With the exception of galactose, labelling patterns did not vary from one morphological stage to another. Galactose, which was localized by using a monoclonal antibody to a galactose-specific lectin purified from insect larval hemolymph, was absent from cell walls of hyphal bodies and conidia but was present on germ-tube and mycelial surfaces. Chitin (N-acetylglucosamine), labelled with a wheat-germ agglutinin-ferritin conjugate, was present in the middle regions of lateral walls and septa, and β1-4 glucans were located in the middle and inner regions, as indicated by binding of a cellulase-gold conjugate. An anti-laminaribiose antibody was used to label β1-3 glucans present in the outer wall areas and inner regions near the plasmalemma. The location of mannose residues as indicated by concanavalin A - ferritin binding was similar to that of the β1-3 glucans; vesicle-like structures were also labelled. None of the probes labelled the outer conidial pellicle or exocellular sheath surrounding germ tubes, and labelling of mycelial sheath was inconsistent. The absence of galactose from Nomuraea hyphal body walls is discussed in terms of host-parasite interaction. Key words: Nomuraea rileyi, entomopathogenic fungi, glycoconjugates, lectins, monoclonal antibodies.

Influence of a negatively charged surface (Teflon disk) on Entomophaga aulicae protoplast morphogenesis under mass fermentation conditions

The effects of a negatively charged surface (Teflon disk) on protoplast morphogenesis for the fungus Entomophaga aulicae under mass fermentation conditions were determined. The control consisted of a vessel lacking such a disk. In the presence of the disk the initial three and sequentially produced protoplast stages (spindle shaped, early fusion sphere, and late fusion sphere protoplasts) recycled with the early fusion sphere predominating. The production of the subsequent and walled stage (i.e., hyphal body) was suppressed. The results are in contrast with those obtained in a previous study using a neutral (Mylar) and a positively charged (polypropylene) disk in which hyphal body production was enhanced. This technique provides a new and subtle approach for altering protoplast developmental patterns which avoids the use of mutagens or added chemical metabolic inhibitors. Key words: Entomophaga aulicae, fungal protoplast morphogenesis, negatively charged surface, mass fermentation.

Enhanced hyphal body production by Entomophaga aulicae protoplasts in the presence of a neutral and a positively charged surface under mass fermentation conditions

The effects of two disks, one with a net neutral charge (Mylar) and one with a positive charge (polypropylene), on hyphal body production from protoplasts of the fungus Entomophaga aulicae under mass fermentation conditions were determined. The results were compared with those obtained with a vessel lacking a disk (control). The presence of either disk greatly enhanced hyphal body production, especially by day 4 of incubation. A maximum level of 94.1% hyphal body production was achieved using the disks as compared to a maximum level of 42% under control conditions. With the exception of aspartic acid, which was produced under control conditions and not utilized when the disks were present, the amino acids were removed from the medium earlier and (or) more completely in the presence of the disks. Glucose utilization and oxygen consumption were greater in the presence of the disks. The utilization of fermentation vessel configurations employing neutral or positively charged disks (surfaces) offers a method for greatly enhancing hyphal body production for biocontrol of the larval stages of forest defoliators. Key words: Entomophaga aulicae, hyphal body production, biological control, mass fermentation, neutral and positively charged surfaces.

Effects of an altered developmental pattern on amino acid uptake for a protoplast isolate of the fungus Entomophaga aulicae under mass-fermentation conditions

The effects of an altered developmental pattern on amino acid uptake for a protoplast isolate of the fungus Entomophaga aulicae were studied under mass-fermentation conditions. Isolate 655A had previously developed through the hyphal body stage, but subsequently lost this ability (then designated I655B) and then developed only as far as the germination of the late fusion sphere stage. This alteration in developmental pattern was accompanied by a shift in the overall amino acid uptake pattern. For comparative purposes, another isolate (I521) which developed only as far as the germination of the late fusion sphere stage was included. The general amino acid uptake patterns fell into three categories and emphasize the similarity of I521 and I655B: (i) earlier and more complete final utilization by isolates 521 and 655B for glutamine, leucine, aspartic acid, threonine, and methionine, (ii) earlier utilization by isolate 655A with more complete final utilization by isolates 521 and 655B for asparagine and proline, and (iii) earlier utilization by isolates 521 and 655A with more complete final utilization by isolates 521 and 655B for serine and tyrosine. The first detectable utilization of serine coincided with late fusion sphere germination for isolates 521 and 655B. During the rapid increase in hyphal body levels for I655A, all added amino acids (except proline, asparagine, and serine which were first utilized earlier) were utilized for the first time. Except for the timing of the initial day of detectable utilization, the pattern of glucose utilization was similar for all three isolates.

Étude en microscopie électronique de Conidiobolus obscurus. I. Formation et germination des azygospores

The multinucleate hyphal bodies of Conidiobolus obscurus are characterized by a striated wall which does not react with Thiéry's stain used to detect free α glycol groups. In the developing mycelial zone, the protoplasm contains microvesicles and electron-dense corpuscles; during ageing of the mycelium, lipid globules varying in size according to the age of the cell and vesicles with dense inclusions are observed in large numbers, whereas the endoplasmic reticulum shows little development. The onset of sporulation is characterized by protoplasmic condensation at a particular point in the hyphal body through successive accumulations of cicatricial partitions. The young prespore is a spherical organ; its wall has the same structure as that of the mycelium, and it contains more or less the same organelles as the hyphal protoplasm. The first sign that the prespore is developing into a spore is the deposition of a very thick inner wall layer (tripartite layer). The inner makeup of the azygospore has all the features of a dormant spore of low metabolic activity having a huge reserve lipid globule. At the onset of germination, the spore wall and the central lipid globule are progressively digested. A new wall entity is synthesized. There is a proliferation of multivesicular and multilaminate bodies. The emergence of the germ tube results from the rupture of the original outer layer of the spore. Results are compared with those observed in various resistance organs of zygomycetes and oomycetes, and discussed in relation to functional metabolic pathways during sporogenesis or germination. [Journal translation]