Comparative analysis of the Metarhizium anisopliae secretome in response to exposure to the greyback cane grub and grub cuticles

Metarhizium anisopliae is a well-characterized biocontrol agent of a wide range of insects including cane grubs. In this study, a two-dimensional (2D) electrophoresis was used to display secreted proteins of M. anisopliae strain FI-1045 growing on the whole greyback cane grubs and their isolated cuticles. Hydrolytic enzymes secreted by M. anisopliae play a key role in insect cuticle-degradation and initiation of the infection process. We have identified all the 101 protein spots displayed by cross-species identification (CSI) from the fungal kingdom. Among the identified proteins were 64-kDa serine carboxypeptidase, 1,3 beta-exoglucanase, Dynamin GTPase, THZ kinase, calcineurin like phosphoesterase, and phosphatidylinositol kinase secreted by M. ansiopliae (FI-1045) in response to exposure to the greyback cane grubs and their isolated cuticles. These proteins have not been previously identified from the culture supernatant of M. anisopliae during infection. To our knowledge, this the first proteomic map established to study the extracellular proteins secreted by M. ansiopliae (FI-1045) during infection of greyback cane grubs and its cuticles.     

Persistence of Conidia of Metarhizium anisopliae in Sugarcane Fields: Effect of isolate and formulation on persistence over 3.5 years

Metarhizium anisopliae is being used in Australia as a biopesticide for control of sugarcane whitegrubs in soil. The field persistence in sugarcane soil of two isolates of M. anisopliae each in four formulations was tested by mixing the formulation with soil which was then placed in PVC rings and buried in sugarcane fields. The two isolates used were FI-1045, M. anisopliae var. anisopliae, the active ingredient in BioCane™ currently used for greyback canegrub control, Dermolepida albohirtum (Coleoptera: Scarabaeidae: Melolonthinae), and FI-147, M. anisopliae var. lepidiotum, being tested as a biopesticide for Lepidiota spp. (Coleoptera: Scarabaeidae: Melolonthinae) and other species of canegrub. The four formulations were rice granules (as in BioCane™), a wettable powder derived from conidia screened from the rice granules, conidia off rice suspended in water and conidia produced on agar plates, dried, and then mixed with water for adding to soil. FI-1045 was tested at three different sites in north Queensland with a range of soil types and climatic conditions while FI-147 was tested at three similarly diverse sites in southern Queensland. The PVC rings were destructively sampled every 6 months for 3.5 years and the number of viable conidia remaining determined by plating onto a selective medium. The exponential decay was determined. Monthly decay rates ranged from 0.0309 to 0.0835 (mean 0.0512). A small proportion of conidia survived the 3.5 years at all sites and all formulations. Overall, isolate FI-147 persisted better than FI-1045, but was used at the more Southerly sites. Rainfall and soil type had negligible effects on persistence. The agar-produced FI-147 conidia declined most slowly, while the two rice-produced but water-formulated conidia gave similar results. Isolate FI-1045 survived best as the BioCane™ formulation and this rice granule formulation was almost as persistent as the agar conidia with FI-147. A small proportion of conidia, in some formulations and at some sites, were recovered from immediately below the rings. This movement was thought to be due to activity of earthworms or mites. The results suggest that 3 years would be the maximum period for a BioCane™ formulation to provide some level of infection in the target pest unless augmented by conidia from infected grubs. The effectiveness of these new conidia may be reduced due to their highly aggregated distribution.     

Resporulation of Metarhizium anisopliae granules on soil and mortality of Tenebrio molitor: Implications for wireworm management in sweetpotato

In Australia, sweetpotato (Ipomoea batatas L.) is vulnerable to root feeding insect pests such as wireworms (e.g., Agrypnus spp.). The number of registered insecticides to control these insect pests is limited and often pest pressure, for example by wireworms, is severe close to harvest, further limiting what insecticides can be applied. Incorporating biological control agents such as entomopathogenic fungi (e.g., Metarhizium anisopliae) into integrated pest management programmes may be feasible in sweetpotato. M. anisopliae has been shown to be effective in controlling more than 200 insects and it is able to reside and grow in the rhizosphere and rhizoplane, suggesting that M. anisopliae could be a promising candidate against soil insect pests. In the study presented here, M. anisopliae was formulated into calcium alginate granules fortified with nutrients. The resporulation of the fungal granules was tested on four different soil types in the laboratory. The biocontrol efficacy of the resulting fungal growth was also examined using larval mealworms, Tenebrio molitor as a model insect in the laboratory and the glasshouse. Our results indicated that sterilised soil favoured optimal fungal resporulation, although different soil types did not have a significant effect on fungal resporulation. The resulting fungal resporulation and growth on sterilised soil caused high mortality (up to 76%) of larval mealworms in the glasshouse, whereas the fungal granules applied to non-sterile soil demonstrated poor resporulation that led to low mortality (13%) of larval mealworms. The result of this study indicates that the manipulation of microbial populations in field soil is required to enhance the fungal growth and potential insect control against wireworms in the field.     

Toxicity of certain insecticides toSturmiopsis inferens,a larval parasite of sugarcane moth borers

Susceptibility of adults and puparia ofSturmiopsis inferens Tns. [Tachinidae, Diptera] to 9 commonly recommended insecticide sprays against sugarcane pests was determined. The chemicals tested as emulsifiable concentrates include lindane 0.1%, endosulfan 0.1%, monocrotophos 0.05%, quinalphos 0.05%, malathion 0.1%, dimethoate 0.1%, cypermethrin 0.01%, fenvalerate 0.01% and decamethrin 0.0014%. Lindane, malathion, dimethoate monocrotophos and quinalphos were highly toxic, while decamethrin had little harmful effect to the adults when exposed for 6 h to filter paper impregnated or sugarcane shoot bits sprayed with the chemicals. However, the insecticides had no harmful effect on the puparia and adult emergence was normal from the puparia sprayed with insecticides. In another study, susceptibility of adults to soil application of lindane EC, carbofuran G, chlorpyriphos G, Sevidol G and whorl application of lindane G, chlorpyriphos G and Sevidol G was tested in pot culture. Except for soil application of lindane EC, all other chemicals had no harmful effect to the adults in pot culture experiment. In a field trial, commonly recommended insecticides against shoot borer,Chilo infuscatellus Snell.viz., soil application of granules of lindane, carbofuran, chlorpyriphos and Sevidol and folia spray of endosulfan did not affect the parasite activity. Institute Publication No 1030.     

Persistence of augmented Metarhiziumanisopliae and Beauveria bassiana in Finnishagricultural soils

The persistence and penetration into soilof surface-applied unformulated conidia of twoisolates of Metarhizium anisopliae and one of Beauveria bassiana at sites with clay, peat andtwo kinds of sand as their soil types and at depthsof from 0 to 20 cm was studied in 1988–1991 underconditions characterized by permanent snow cover andfrozen soil in the winter time. At 0–5 cm depth, M. anisopliae persisted throughout the experiment atall sites, clay being the most and peat the leastfavourable soil for persistence. Clay and one of thesandy soils were the least and peat the most conducivesoil to penetration of M. anisopliae from thesurface to deeper soil layers and persistence therein.Differences in persistence were evident between thetwo M. anisopliae isolates in the sandy and peatsites, but not in the clay site. Three yearspostapplication there were still enough infectiouspropagules of M. anisopliae in soil of allsites to infect over 80% of the Tenebriomolitor larvae used as baits in samples taken fromthe cylinders of all soils. All the augmentedpropagules of B. bassiana disappeared duringthe first winter after application in clay and one ofthe two sandy sites, but some persistence one yearpostinoculation was evident at 0–5 depth in one ofthe sand soils and at 0–5 and 5–10 cm depths in peat.     

Virulence of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> using soybean oil formulation for control of the cotton stainer bug, <Emphasis Type="Italic">Dysdercus peruvianus</Emphasis>

The cotton stainer bug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) is an insect pest that causes heavy losses in cotton plantations. The need to reduce the use of insecticides for control of this pest has increased steadily, and Metarhizium anisopliae (Ascomycota: Clavicipitaceae) could be an important biopesticide candidate to control this pest. The effect of M. anisopliae on D. peruvianus nymphs and adults using formulations with soybean oil and Agral^® was evaluated. Formulation using 10% soybean oil added to 10⁸ conidia mL^?1 (grown on used and reused rice) was the most effective for nymph and adult, causing 100% mortality 6 and 7 days after exposure, respectively. The SEM analysis of infected insects showed that M. anisopliae conidia were able to adhere anywhere on the exoskeleton, but were more abundant between the joints. Using the same rice for two batches of growth may be an option for improving commercial conidial production of M. anisopliae and may reduce overall costs. Its effect on D. peruvianus adults opens a new possibility for using this fungus as an alternative to chemical pesticides and the use of M. anisopliae in association with integrate pest management.     

Efficacy of Metarhizium anisopliae microsclerotial granules

The entomopathogenic fungus Metarhizium anisopliae has very recently been shown to produce microsclerotia (MS) – compact, heavily melanised, hyphal aggregates – in liquid media. Soil incorporation bioassays of dried MS preparations of three isolates of M. anisopliae were conducted using third instar Tetanops myopaeformis (sugarbeet root maggot) in clay and/or clay loam field soils as a model system to demonstrate efficacy. At rates as low as 23 mg MS granules/100 g dry soil, the biocontrol efficacy of MS granules of M. anisopliae Strain F52 produced in liquid media with a high carbon concentration (36 g/L) and high C:N ratios (30:1, 50:1) were superior to MS preparations produced in low carbon (8 g carbon/L) media and a high carbon medium with a 10:1 C:N ratio. Bioassays using MS formulations of M. anisopliae strains MA1200 and TM109 produced in high carbon and high C:N ratio media were superior in efficacy to the other MS production media tested. MS preparations of M. anisopliae F52 showed superior efficacy against the sugarbeet root maggot in comparison with more conventional, conidia-covered nutritive (corn grit) granules in a clay and clay soil. The MS granules were also highly efficacious against the sugarbeet root maggot at soil moisture levels as low as 0.983 A _w (?2.33 MPa). Granular preparations incorporating Metarhizium MS can serve as a viable formulation for the use of this fungus against soil insects.     

Impact of fungicides on Metarhizium anisopliae in the rhizosphere, bulk soil and in vitro

The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) is registered in the United States and The Netherlands for black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) control in container-grown ornamentals. These studies were conducted to determine the compatibility of M. anisopliae (F52) with a wide range of fungicides commonly applied to container-grown ornamentals for the management of soil-borne plant pathogens. The impact of fungicides on spore germination and mycelial growth were determined in vitro. In addition, M. anisopliae persistence in bulk and rhizosphere soil was determined 30 days following dual application of each fungicide at 7–28 days intervals as prescribed. A number of fungicides (thiophanate-methyl, dimethomorph, captan, triflumizole, triflozystrobin, pyraclostrobin, azoxystrobin) inhibited spore germination in vitro. A larger number of fungicides (fosetyl-AI, thiophanate-methyl, dimethomorph, captan, quintozene, triflumizole, fludioxanil, triflozystrobin, pyraclostrobin, fludiox-mefanox, iprodione, azoxystrobin, phosphorus acid/K-salts) inhibited mycelial growth in vitro. Only three fungicides (etridiazole, propamocard and mafanoxam) had no significant impact in vitro on spore germination or mycelial growth. While a number of fungicides had a detrimental impact in vitro, there was no impact on M. anisopliae populations in bulk soil following dual application of any fungicide. However, the fungicides captan and triflumizolet, which have a short reapplication interval, had a detrimental impact on M. anisopliae populations in the rhizosphere. As researchers develop rhizosphere competence as an alternative management strategy for black vine weevil, the fungicides captan and triflumizole should be avoided.     

Economic injury levels for the scarabaeid Maladera matrida infesting peanut fields in Israel

Control of Maladera matrida Argaman (Coleoptera: Scarabaeidae) in peanut fields in the Negev region, Israel, relies on the `rule of thumb' use of insecticides. The aim of this study was to develop an economic injury level (EIL) for a more judicious application of insecticides. Field studies were conducted from 1991 to 1993 to determine the relationship between the cumulative number of different immature stages and the damage of peanuts (Arachis hypogaea L.). Observed damage to peanut yield was found to relate best to second- and third-instars (white grubs). This was supported by grub infestation experiments on potted peanut plants. Three different EIL levels for second- and third-instars of M. matrida were established in connection with the peanut plant developmental stages as follows: an average of 13 grubs per soil sample (25 by 25 by 60 cm) taken during the first 3 weeks of nut development, of 4 grubs per soil sample during week 4 to 6, and of 1.5 grubs per soil sample from week 7 until harvest.     

Lethal and trans-generational effects of Metarhizium anisopliae on red palm weevil,Rhynchophorus ferrugineus,using age-stage,two-sex life table

Outbreaks of red palm weevil, Rhynchophorus ferrugineus Olivier, in Pakistan are considered a very serious threat to the date industry, owing to the sudden death of date palm trees. Current integrated management strategy largely depends on insecticides, with negative impacts on the environment. Integration of bio-control strategies incorporating entomopathogenic fungi (EPF) may provide an ecologically safe alternative. In the current study the effectiveness of Metarhizium anisopliae Sorokin against R. ferrugineus was investigated using the immersion method and the effects of this fungus on biological parameters of R. ferrugineus were estimated by age-stage, two-sex life table software. After treatment with M. anisopliae isolate (Ma-M₂), lethal concentration (LC₅₀) 1.2 × 10⁶ spores/mL was determined against R. ferrugineus 3rd instar grubs. M. anisopliae infected grubs showed reduced net reproductive rate R_o (75.55) and mean generation time T (113.886 days) and intrinsic rate of increase r (0.03797) compared to the untreated group. Also, adult longevity of males and females were significantly reduced in the M. anisopliae-infected group (F₁) as compared to the untreated group. Trans-generational studies revealed that grubs stage duration (69.34 days) and fecundity (181.32 eggs) of the M. anisopliae-infected group (F₁) were decreased compared to the untreated group. The use of lasting and adverse effects on the biological parameters of R. ferrugineus, indicate that treatment with EPF may provide an eco-friendly management strategy for this pest.     

Production of α-galactosidase by Monascus grown on soybean and sugarcane wastes

Extracts of both sugarcane and soybean wastes supported the growth of Monascus but sugarcane waste was superior for the production of -galactosidase. An aqueous extract prepared from 5% (w/v) soybean waste and 7% (w/v) sugarcane waste gave the best result and was superior to the standard peptone/glucose/yeast extract medium. Liquid-solid mixtures were slightly less effective. Enzyme production could be enhanced by adding raffinose. Enzymatic hydrolysis of p-nitrophenyl--D-galactoside was optimal at pH 4.5. Raffinose and stachyose were hydrolysed to sucrose and galactose.     

Biodegradation of beta-cyfluthrin by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain S1

-Cyfluthrin [-cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate] pesticide has been in agricultural use in the recent years for controlling Lepidopteran pests affecting solanaceous crops. The extensive use of synthetic pyrethroids like -cyfluthrin has resulted in wide spread environmental contamination. The purpose of this study was to isolate bacteria from soil and to determine their ability to degrade -cyfluthrin and identify the intermediates in culture broth using spectroscopy. An aerobic bacterium capable of degrading -cyfluthrin was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain S1) had 100% identity to the sequence from Pseudomonas stutzeri. Finally products formed during degradation of -cyfluthrin have been identified as -cyano-4-fluoro-3-phenoxybenzyl-3(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (M.W. 341); 4-fluoro-3-phenoxy--cyanobenzyl alcohol (M.W. 243) and 3(2,2-dichlorovinyl)-2,2-dimethyl cyclopropanecarboxylic acid (M.W. 208).     

FI-1045: A Profile of a Commercially Useful Isolate of Metarhizium anisopliae var. anisopliae

The isolate FI-1045 is the basis of a mycoinsecticide, BioCane TM granules recently registered for the control of greyback canegrub, Dermolepida albohirtum (Coleoptera: Scarabaeidae: Melolonthinae) in Australian sugarcane fields. The isolate was obtained from a naturally infected larva of D. albohirtum collected from Tully in north Queensland. The isolate can be distinguished from others infecting the same insect and also other species of canegrub by means of RAPD patterns and sequence data from the ITS region. A comparison of a stored FI-1045 isolate with three derived isolates which had different histories of host-passage, showed no variation in RAPD pattern. All isolates grew well at temperatures between 20 and 30°C but did not grow at 35°C and grew slowly at 15°C. However, on potato dextrose agar, the original FI-1045 grew more rapidly and did not produce as much pigment as the derived isolates. It is speculated that this difference was due to the storage method used with the original FI-1045 being stored at -70°C and the other isolates being freeze-dried. Bioassays against third instar greyback canegrubs gave a mean LC 50 of 8.7 ×10 4 conidia g -1 peat substrate after 10 weeks. Using Tenebrio molitor as a host, it was found that conidia taken directly from the infected insect were similar in virulence to the cultured FI-1045. Using injection of culture filtrate as the assay method, it was found that FI-1045 produced destruxins. In laboratory host range tests, a dose of 10 6 conidia g -1 peat killed 96% of southern oneyear canegrubs, Antitrogus consanguineius , 85% of Lepidiota picticollis and less than 30% of the other five species of canegrub tested.     

Entomopathogenic fungi as a potential control agent against the lesser mealworm, <Emphasis Type="Italic">Alphitobius diaperinus</Emphasis> in broiler houses

We tested the pathogenicity of 18 Metarhizium anisopliae (Metschn.) Sorokin isolates and 22 Beauveria bassiana (Balsamo) Vuillemin isolates against Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) larvae and adults. The efficacy of the most virulent isolate—M. anisopliae K—was evaluated in containers with a concrete bottom covered with wood shavings, under simulated poultry house conditions. Application of conidia of this isolate to the shavings or directly to the concrete bottom reduced the yield of larvae in 8–15 time compared with the control. In another test, the mortality of mature larvae placed on previously inoculated shavings or bottom reached 80–90% within 14 days, compared with 14% in the control. The residual activity of conidia kept at 28°C retained its initial level during 14 days post-inoculation, but declined after three weeks. Based on our data M. anisopliae has considerable potential for the control of A. diaperinus.

Evaluation of entomopathogenic fungi as potential biological control agents of the dengue mosquito,Aedes aegypti (Diptera: Culicidae)

Dengue is a global health concern. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control, so alternative tools are urgently needed. One approach is the use of biopesticides comprising entomopathogenic fungi, e.g., Beauveria bassiana and Metarhizium anisopliae. These fungi may decrease disease transmission by reducing mosquito vector longevity and also occur worldwide, although many isolates have not been tested for virulence against mosquitoes. Ninety-three isolates of entomopathogenic fungi representing six species (B. bassiana, M. anisopliae, Isaria fumosorosea, I. farinosa, I. flavovirescens, and Lecanicillium spp.) were screened as potential biological control agents of Aedes aegypti. A hierarchical, multi-criteria experimental design was undertaken to find suitable isolates. Initial screening was performed via in vitro assays measuring radial growth and spore persistence, eliminating isolates with poor growth or viability on nutrient-rich substrate. Subsequent measurements of spore persistence revealed that only nine of 30 strains tested had half-lives exceeding 3 weeks. Ten isolates were chosen for in vivo bioassays against adult Ae. aegypti. From these assays, two Australian isolates of B. bassiana, FI-277 and FI-278, appeared to be most promising. Both isolates were shown to be virulent against Ae. aegypti at 20, 26, and 32°C. Spreading spores manually onto substrate was found to be more efficacious than spraying. Ae. aegypti infected by manually-spread spores on cotton substrate were found to have an LT₅₀ of 3.7±0.3 days. These characteristics suggest that FI-277 has promise as a dengue mosquito biocontrol agent, either alone or combined with conventional chemical insecticides.     

A phase-contrast and immunofluorescence study of adrenal medullary chromaffin cells in culture: Neurite formation,actin and chromaffin granule distribution

Summary Immunofluorescence studies of bovine chromaffin cells in culture with specific antibodies against dopamine-gb-hydroxylase gave a distinct punctate pattern of labelling, reflecting the distribution of chromaffin granules. There was strong staining of cell extensions and growth cones. Linear arrays of fluorescent dots were observed, suggesting an association of granules with a filamentous cytoskeleton. Labelling of neuritic processes was periodic, perhaps indicative of a packaging of secretory granules.Chromaffin cells stained strongly with specific anti-actin antisera. Fine filament bundles were observed, and also diffuse staining, some punctate labelling and staining of the plasma membrane or sub-membranous cytoplasm. Growth cones and non-terminal cytoplasmic varicosities contained significant amounts of actin. Colchicine (5×10^-5M) caused retraction of neuritic extensions and formation of lateral growth cones. Cytochalasin (10g/ml) caused ballooning of terminal growth cones and non-terminal cytoplasmic varicosities. Phalloidin (10^-4M) stimulated microspike formation. The results are discussed in terms of the role of the cytoskeleton in growth cone formation, cell-substratum contacts and the transport of chromaffin granules.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Methods for studying the nematophagous fungus Verticillium chlamydosporium in the root environment

In order to exploit fully the biocontrol potential of the nematophagous fungus Verticillium chlamydosporium, it is important to understand the ecology of the fungus in soil, and interactions with both plant and nematode hosts. Several approaches for studying the fungus in soil and the root environment are compared. These include a semi-selective medium for V. chlamydosporium, PCR primers specific for the fungal -tubulin gene, and monoclonal antibodies. In addition to providing a target for species-specific primers, the -tubulin gene is implicated in resistance to the fungicides used in the semi-selective medium, and the genetic basis for this is investigated. Culture and PCR-based methods were used to screen for the presence of the fungus in field soils known to have been suppressive to cereal cyst nematode and that contained V. chlamydosporium populations. Monoclonal antibodies specific for either hyphae or conidia of the fungus were obtained, and their application as a tool for visualising the infection process on the root was explored.     

Conversion of free β-amylase to bound β-amylase on starch granules in the barley endosperm during desiccation phase of seed development

Summary Association of -amylase with starch granules in the starchy endosperm of barley (Hordeum vulgare L. cv. Menuet) grains was characterized biochemically. In whole homogenates of dry seeds, two forms of -amylase were detected: one is free -amylase extractable with saline solution and the other is bound -amylase extractable with saline solution containing a reducing agent. The two forms of -amylase were shown to be identical in terms of mobility on disc gels, antigenicity, and molecular specific activity, indicating that the -amylase molecules of the two forms are identical. The starch granules were isolated from either dry seeds or mature seeds harvested before the desiccation phase. Both starch granule preparations were morphologically identical by microscopic inspection. The bound -amylase was predominantly associated with starch granules isolated from dry seeds, whereas it was not associated with starch granules from mature seeds harvested before desiccation. Overall results show that the periphery of starch granules is the major site of deposition for bound -amylase in dry seeds. The association of -amylase with starch granules occurs during the desiccation phase of seed development, resulting in the conversion of free -amylase into a bound form.Recipient of an award from the Union Générale de la Brasserie Française (I. H.-N.) and from the Centre National de la Recherche Scientifique and the Japan Society for the Promotion of Science under the France-Japan Cooperative Science Programme, 1985 (M.N.).