Effect of prolonged storage of spores on field applications of Nosema locustae (Microsporida: Nosematidae) against grasshoppers

Spores of Nosema locustae that were freshly prepared, stored in water at ?10°C for 8 months to 3 years, or stored in cadavers for 1 year at ?10°C were applied on bran at the rate of 10⁹ spores/1.5 lb bran per acre. Applications of fresh spores resulted in higher density reductions and higher incidence of infection among survivors than applications of spores stored in water or cadavers in both a complex of grasshoppers predominated by a single species and in a mixed species complex. Density reductions due to treatment with fresh spores were similar in the two populations, but the mixed species complex had a lower incidence of infection than the complex in which one species predominated. Applications of fresh spores reduced grasshopper densities in both complexes to levels below the economic thresholds.     

Reduction of grasshopper populations following field application of the fungus Beauveria bassiana

Conidia of the Hyphomycete fungus Beauveria bassiana (Bals.) were applied in an attempt to reduce field populations of grasshoppers, primarily the migratory grasshopper Melanoplus sanguinipes (Fabricius). Dry spores were applied with wheat bran carrier to three fallow fields at a rate of 2.0 × 10¹³ spores ha^?1 in 10 kg bait ha^?1. Examination of culture plates that had been placed in the field to capture spores and of bran carrier with scanning electron microscopy indicated that a substantial portion of the B. bassiana colony—forming units (spores and clumps of spores) did not adhere to the bran and were applied in the field as free particles. Grasshoppers collected from the treated plots at intervals after application were assayed for infection by B. bassiana. The observed rate of mycosis in the treated populations was 70% of those collected after 2 days, declining to 41% by 13 days and 5% by 19 days after application. Analysis of reductions in population density gave results in agreement with the infection data. Treated populations declined 60% and 33% by 9 and 15 days after application respectively. The reductions were significant on both post—treatment sampling dates (p < 0.05) and the three replicated fields gave comparable results. This is the first field demonstration of effectiveness of this fungus as a microbial control agent of grasshoppers.     

Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore properties

Aims: To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results: The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores’ inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50–150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l‐PG) synthesis exhibited a 30–50% decrease. Spore sensitivity to H₂O₂ and tert‐butylhydroperoxide was increased 30–60% in the absence of the major CL synthase, but these spores’ sensitivity to NaOCl or Oxone^? was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l‐PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10‐fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions: Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore’s inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study: The results of this study provide insight into roles of inner membrane lipids in spore properties.     

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.     

Infection of the corn earworm, Heliothis zea, with Nosema acridophagus and Nosema cuneatum from grasshoppers: Relative virulence and production of spores

Per os inoculations of 4- to 6-day-old larvae of the corn earworm, Heliothis zea, with suspensions containing 10⁶ spores of Nosema acridophagus or 10⁴, 10⁵, and 10⁶ spores of Nosema cuneatum retarded the growth and development of the larvae. Migratory grasshoppers, Melanoplus sanguinipes, inoculated with N. acridophagus produced fewer spores than similarly inoculated corn earworms, but spore production was similar in these insects when they were inoculated with N. cuneatum. Standard bioassay procedures showed that spores of both microsporidians were some-what more virulent when they were produced in corn earworms than when they were produced in grasshoppers. Spores of these microsporidians might be produced more efficiently in corn earworm larvae than in grasshoppers.     

Effect of spore concentration on germination and autotropism inTrichoderma hamatum

Summary The effect of spore concentration on spore germination and germtube growth ofTrichoderma hamatum on water agar and on potato dextrose agar (PDA) was studied. Increasing inoculum size up to 10⁹ spores/plate on PDA and up to 10⁷ spores/plate on water agar shortened the incubation period required for germtubes emergence and increased germination rate. However, on water agar germination was inhibited at 10⁸ and was completely arrested at 10⁹ spores/plate. Inhibition in germination of 10⁷ spores/plate was observed on water agar when the plates were preincubated with 10⁹ spores/plate for 5 h or more. Addition of glucose and ammonium nitrate to the water agar medium allowed only 25% of the spores to germinate at 10⁹ as compared to 78% at 10⁷ spores/plate after 8 h of incubation. Addition of polysaccharides to the C+N supplemented medium, significantly increased germination up to 84% as compared to 100% on PDA, after 8 h of incubation. Germlings ofTrichoderma hamatum phialospores exhibited positive autotropism and anastamosis on both media. The phenomenon was positively related to inoculum size, being most pronounced at 10⁷ spores/plate.     

Diversity of arbuscular mycorrhizal fungi across a fragmented forest in Panama: insular spore communities differ from mainland communities

It is now understood that alterations in the species composition of soil organisms can lead to changes in aboveground communities. In this study, we assessed the importance of spatial scale and forest size on changes in arbuscular mycorrhizal fungal (AMF) spore communities by sampling AMF spores in soils of forested mainland and island sites in the vicinity of Gatun Lake, Republic of Panama. We encountered a total of 27 AMF species or morphospecies, with 17, 8, 1 and 1 from the genera Glomus, Acaulospora, Sclerosystis, and Scutellospora, respectively. At small scales (<100 m²), we found little evidence for spatial structuring of AMF communities (decay of Morisita-Horn community similarity with distance). However, at large spatial scales, we found that the AMF spore community of a mainland plot was more similar to other mainland plots several kilometers (>5) away than to nearby island plots (within 0.7 km). Likewise, most island plots were more similar to other island plots regardless of geographic separation. There was no decay in AMF species richness (number of species), or Shannon diversity (number of species and their spore numbers) either with decreasing forest-fragment size, or with decreasing plant species richness. Of the six most common species that composed almost 70% of the total spore volume, spores of Glomus tsh and G. clavisporum were more common in soils of mainland plots, while spores of Glomus small brown and Acaulospora mellea were more abundant in soils of island plots. None of these common AMF species showed significant associations with soil chemistry or plant diversity. We suggest that the convergence of common species found in AMF spore communities in soils of similar forest sizes was a result of forest fragmentation. Habitat-dependent convergence of AMF spore communities may result in differential survival of tree seedlings regenerating on islands versus mainland.     

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).     

Optimization of the spore production of Penicillium roqueforti in solid substrate fermentation on buckwheat seeds

Summary The effect of substrate (buckwheat seeds) pretreatment on the growth and the sporulation behaviour of Penicillium roqueforti is presented. When a saccharifying enzyme (-amylase) is added to a medium which exhibits a low water content (0.46 g water/g initial dry matter, IDM), a more rapid internal colonization of the seeds occurs, but the final spore production does not increase and remains close to 8.10⁹ spores/g dry matter (DM) at 500 h. No carbon source limitation is then observed. The addition of casein hydrolysate to this medium gives rise to a great increase of the sporulation, since 14.5 10⁹ spores/g DM are obtained after 600 h. This result is attained by a better spore yield from the mycelium, the substrate colonization being unchanged. High water content (0.60 g water/g IDM) of buckwheat seeds induces a shorter cultivation time along with a higher biomass production. However, the spore content of the medium remains close to the low water content one, but 60% total spores are external against 30% to 35% in the other media.     

Effect of radioprotective agents in sporulation medium on Bacillus subtilis spore resistance to hydrogen peroxide, wet heat and germicidal and environmentally relevant UV radiation

Aims: To determine the effects of cysteine, cystine, proline and thioproline as sporulation medium supplements on Bacillus subtilis spore resistance to hydrogen peroxide (H₂O₂), wet heat, and germicidal 254 nm and simulated environmental UV radiation. Methods and Results: Bacillus subtilis spores were prepared in a chemically defined liquid medium, with and without supplementation of cysteine, cystine, proline or thioproline. Spores produced with thioproline, cysteine or cystine were more resistant to environmentally relevant UV radiation at 280–400 and 320–400 nm, while proline supplementation had no effect. Spores prepared with cysteine, cystine or thioproline were also more resistant to H₂O₂ but not to wet heat or 254‐nm UV radiation. The increases in spore resistance attributed to the sporulation supplements were eliminated if spores were chemically decoated. Conclusions: Supplementation of sporulation medium with cysteine, cystine or thioproline increases spore resistance to solar UV radiation reaching the Earth’s surface and to H₂O₂. These effects were eliminated if the spores were decoated, indicating that alterations in coat proteins by different sporulation conditions can affect spore resistance to some agents. Significance and Impact of the Study: This study provides further evidence that the composition of the sporulation medium can have significant effects on B. subtilis spore resistance to UV radiation and H₂O₂. This knowledge provides further insight into factors influencing spore resistance and inactivation.     

Trichostomopsis aaronis (Lor.) Agnew & Townsend new to the U.S.S.R.

Abstract

Occurrence of Ptilidium pulcherrimum in transects and spore dispersal from a single colony have been studied in a coastal spruce forest in northern Sweden. The main substrate type was rotting wood with 75% of all occurrences. Annual spore production was 68,500 spores/m² forest, 640,000 spores/m² substrate and 44,000,000 spores/m² colony. Almost 50% of the spores were deposited within 2.5 m of the colony. Annual spore deposition between colonies was estimated to be between 24,000–39,000 and deposition on the main substrate, decaying logs, was about 340–600 spores/m² forest. P. pulcherrimum showed a clumped distribution pattern up to about a 15 m neighbourhood distance. This pattern could not be explained by a similar clumping of the substrate. Instead a limitation by distance in establishment due to a deficit of spores is assumed.     

蝗虫微孢子虫病在草原蝗虫优势种种群及空间的分布

在采用蝗虫微孢子虫Nosema locustae防治过的草场中进行抽样调查,研究了草原蝗虫优势种类、混合种群平均密度与蝗虫微孢子虫疾病分布之关系,以及该疾病的空间分布。在防治后的当年,蝗虫微孢子虫疾病的感染率随着混合种群平均密度及靶标蝗虫亚洲小车蝗Oedaleus asiaticus的感病率的下降而降低。但是,次靶标蝗虫如宽须蚁蝗Myrmeleotettixpalpalis(一种中后期发生的种类)其感病率呈上升趋势,表明该疾病可在不同发生期种类蝗虫之间进行有效地传播。病蝗虫在防治后第7d其空间分布呈随机分布(Poisson),第28d 则是聚集分布,第40d时也呈聚集分布。于1993年、1994年对1988年(样区Ⅱ)、1989 年(样区Ⅲ)采用微孢子虫防治过的草场进行抽样调查。结果表明,在二个样区中,二年混合种群平均虫口密度与混合种群的平均感病率呈正相关(相关系数分别为r=0.289, r=0.479)。蝗虫微孢子虫病在主要优势种,如亚洲小车蝗、宽须蚁蝗、白边痂蝗Bryode maluctuosumluctuosum、皱膝蝗Angaracris /I>spp.、毛足棒角蝗Dasyhippus barbipes均有分布。二个样区中的混合蝗虫种群的平均感病率在1994年显著低于1993年。混合蝗虫种群的种类组成也有所变化,与1993年相比,1994年宽须蚁蝗及白边痂蝗的比例上升较大,而亚洲小车蝗的比例下降。经过5—7年的扩散,蝗虫微孢子虫病至少可扩散距防治区1 000m,其扩散方向可能与风及地势等有关。     

Verticillium wilt of the hop: field studies on wilt in a resistant cultivar in relation to nitrogen fertilizer applications

In two large-scale observation trials on commercial farms inorganic nitrogen fertilizer was applied at 70 lb. N/acre (N1), 1401b. N/acre (N2) and at a normal commercial rate of 210 lb. N/acre (N3). Mean wilt incidence was 60 and 25% less with Ni and N2 respectively than with N3 during 5 years at one site and 3 years at the other. During these periods wilt declined progressively and this was tentatively attributed to the reduced intensity of host colonization, and to progressive declines in soil infectivity resulting from effects on the quantity, quality and longevity of annually produced inoculum. Marked annual fluctuations of wilt incidence and yields occurred at one site and these were associated with weather in the spring and early summer: wilt incidence was inversely related to soil temperature, and yield was directly related to rainfall. Yields were not diminished by the low-N treatments, but clear correlations between yields and wilt incidence were probably obscured by the differential effects of weather conditions at the three levels of N application. The results emphasized the importance of reducing traditionally high N applications and, on farms where fluctuating wilt is severe, of applying the minimum levels of N commensurate with the maintenance of satisfactory yields.     

The effects on soil fauna of insecticides tested against wireworms (Agriotes spp.) in wheat

The organophosphorus insecticides Bayer 38156 (O-ethyl S-p-tolyl ethyl phosphonodithioate), trichloronate, Stauffer N 2790 (O-ethyl S-phenyl ethyl phosphonodithioate), thionazin and fenitrothion were compared with aldrin, dieldrin and γ-BHC for their effects on soil fauna, particularly wire-worms, and on crop yields in 1964 and 1965. At 1·5 lb active ingredient/acre, none of the organophosphates had as great an effect on wireworms as an aldrin spray at 2·25 lb a.i./acre or a dieldrin seed dressing at 2·25 lb a.i./acre. Some treatments significantly increased and some significantly decreased numbers of mites and Collembola. Except for Allolobophora chlorotica in plots treated with Bayer 38156, earthworm numbers were greater in plots sprayed with Bayer 38156 or aldrin, or sown with dieldrin-dressed seeds, than in untreated plots. In May 1964, one month after sowing, untreated plots had significantly fewer plants than plots sprayed with aldrin, trichloronate or Bayer 38156, or sown with γ-BHC or dieldrin-dressed seeds, but yields from untreated plots at harvest were high for such a large wireworm population and did not differ significantly from yields of treated plots in either year. The persistence of thionazin and Bayer 38156 in treated plots was measured by a bioassay using Collembola. Bayer 38156 was detected in plots 1 month after spraying but not after 6 months. Thionazin left detectable residues 1 month after spraying in the two acid plots but not in the two alkaline plots. More frequent samples taken from thionazin-treated plots in 1965 showed a similar pattern of persistence, and laboratory tests, using soil mixed with various amounts of powdered calcium carbonate, confirmed that thionazin persisted longer in more acid soils.     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Dispersal of Septoria nodorum Pycnidiospores by Simulated Rain and Wind

The influence of wind on the splash dispersal of Septoria nodorum pycnidiospores was studied in a raintower/wind tunnel complex with single drops or simulated rain falling on spore suspensions or infected stubble with windspeeds of 1.5 to 4 m/sec. When single drops fell on spore suspensions (depth 0.5 mm, concentration 7.8 × 10⁵ spores/ml) most of the spore-carrying droplets collected on fixed photographic film between 0–4 m downwind (windspeed 3 m/sec) were >200 μm in diameter. However, most spores were carried in droplets with diameter > 1000 μm, 70 % of which carried more than 100 spores. When simulated rain fell on infected stubble most of the spore-carrying droplets collected beyond 1 m downwind (windspeeds 1.4 and 4 m/sec) were <200 μm in diameter and none were >600 μm; most of these droplets carried only one spore. The distribution of splash droplets (with diameter >100 μm) deposited on chromatography paper showed a maximum at 40–50 cm upwind of the target but many more droplets were deposited 20–30 cm downwind, when single drops fell on a spore suspension (concentration 1.2 × 10⁵ spores/ ml) containing fluorescein dye with a windspeed of 2 m/sec; droplets were collected up to 3 m downwind but not more than 70 cm upwind. With a windspeed of 3 m/sec, numbers of sporecarrying droplets and spores collected on film decreased with increasing distance downwind; most were collected within 2 m of the target but some were found up to 4 m. When simulated rain fell on infected stubble, increasing the windspeed from 1.5 to 4 m/sec greatly increased the number of spores deposited more than 1 m downwind. At 1.5 m/sec none were collected beyond 2 m downwind, whereas at 4 m/sec some were collected at 4 m. A few air-borne S. nodorum spores were collected by suction samplers at a height of 40 cm at distances up to 10 m downwind of a target spore suspension on which simulated rain fell.     

Verticillium lecanii Spore Formulation Using UV Protectant and Wetting Agent and the Biocontrol of Cotton Aphids

Verticillium lecanii spores (10⁸ spores ml⁻¹) suspended in 1% (w/v) montmorillonite SCPX-1374 and 1% (w/v) of the wetting agent, EM-APW#2, which is a polyoxyethylene, had approx. 80% survival after exposure to UV-C for 30 min and about 93% after exposure to UV-B for 6 h. In greenhouse testing, cotton aphid densities increased 14-fold over their initial density in 15 d without spore application. However, initial cotton aphid densities were decreased by 60% of the initial level when plants were treated with the spore formulation.     

Moist-heat resistance, spore aging, and superdormancy in Clostridium difficile

Clostridium difficile spores can survive extended heating at 71°C (160°F), a minimum temperature commonly recommended for adequate cooking of meats. To determine the extent to which higher temperatures would be more effective at killing C. difficile, we quantified (D values) the effect of moist heat at 85°C (145°F, for 0 to 30 min) on C. difficile spores and compared it to the effects at 71 and 63°C. Fresh (1-week-old) and aged (≥20-week-old) C. difficile spores from food and food animals were tested in multiple experiments. Heating at 85°C markedly reduced spore recovery in all experiments (5 to 6 log(10) within 15 min of heating; P < 0.001), regardless of spore age. In ground beef, the inhibitory effect of 85°C was also reproducible (P < 0.001), but heating at 96°C reduced 6 log(10) within 1 to 2 min. Mechanistically, optical density and enumeration experiments indicated that 85°C inhibits cell division but not germination, but the inhibitory effect was reversible in some spores. Heating at 63°C reduced counts for fresh spores (1 log(10), 30 min; P < 0.04) but increased counts of 20-week-old spores by 30% (15 min; P < 0.02), indicating that sublethal heat treatment reactivates superdormant spores. Superdormancy is an increasingly recognized characteristic in Bacillus spp., and it is likely to occur in C. difficile as spores age. The potential for reactivation of (super)dormant spores with sublethal temperatures may be a food safety concern, but it also has potential diagnostic value. Ensuring that food is heated to >85°C would be a simple and important intervention to reduce the risk of inadvertent ingestion of C. difficile spores.     

Destruction of Spores on Building Decontamination Residue in a Commercial Autoclave

The U.S. Environmental Protection Agency conducted an experiment to evaluate the effectiveness of a commercial autoclave for treating simulated building decontamination residue (BDR). The BDR was intended to simulate porous materials removed from a building deliberately contaminated with biological agents such as Bacillus anthracis (anthrax) in a terrorist attack. The purpose of the tests was to assess whether the standard operating procedure for a commercial autoclave provided sufficiently robust conditions to adequately destroy bacterial spores bound to the BDR. In this study we investigated the effects of several variables related to autoclaving BDR, including time, temperature, pressure, item type, moisture content, packing density, packing orientation, autoclave bag integrity, and autoclave process sequence. The test team created simulated BDR from wallboard, ceiling tiles, carpet, and upholstered furniture, and embedded in the BDR were Geobacillus stearothermophilus biological indicator (BI) strips containing 10⁶ spores and thermocouples to obtain time and temperature profile data associated with each BI strip. The results indicated that a single standard autoclave cycle did not effectively decontaminate the BDR. Autoclave cycles consisting of 120 min at 31.5 lb/in² and 275°F and 75 min at 45 lb/in² and 292°F effectively decontaminated the BDR material. Two sequential standard autoclave cycles consisting of 40 min at 31.5 lb/in² and 275°F proved to be particularly effective, probably because the second cycle's evacuation step pulled the condensed water out of the pores of the materials, allowing better steam penetration. The results also indicated that the packing density and material type of the BDR in the autoclave could have a significant impact on the effectiveness of the decontamination process.     

Fecundity,spore recruitment and size in Gelidium sesquipedale (Gelidiales,Rhodophyta)

Gelidium sesquipedale fecundity was quantified by counting tetrasporangial sori and cystocarps per meter squared and by estimating the number of spores contained inside them. These were obtained by regression on a size metric of reproductive structures. Tetrasporangial sori length and cystocarp thickness were the best estimators of spore number. To assess spore recruitment, 12 pottery tiles were fixed to the bottom, and the appearance of small fronds was monitored.No clear seasonal pattern of reproduction was found. Tetraspore production peaked in March 1990 with 10.4 × 10⁶ spores m^–2, whereas the carpospore peak was lower, 4.9 × 10⁵ spores m^–2 in July 1989. Recruitment followed tetraspore peaks. The probability of a G. sesquipedale tetraspore making the transition to a recruit was 4.7 × 10^–5. Frond length was significantly related to tetrasporangial sori number, while cystocarp number was only related to frond branching order. Minimum size for reproduction was 6.9 cm for gametophytes and 5.4 cm for tetrasporophytes; very rarely were cystocarpic fronds smaller than 9 cm, while tetrasporic fronds were often longer than 15 cm. Cystocarpic fronds were significantly shorter and had more branches than tetrasporic fronds.