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Histology of V—A mycorrhizal development in guayule seedlings
831
Mycol. Res. 94 ( 6 ) :831-834 (1990) Printed in Great Britain
Histology of V-A mycorrhizal development in guayule seedlings
A. J. VIETTI A N D J. VAN STADEN UN/FRD Research Unit for Plant Growth and Development, Department of Botany, University of Natal, P 0 Box 375, Pietemritzburg 3200, Republic of South Africa
Histology of V-A mycorrhizal development in guayule seedlings. Mycological Research
94 ( 6 ) :831-834 (1990).
Guayule seed was germinated and grown in acid-washed quartz sand seeded with inoculum of the vesicular arbuscular mycorrhizal (VAM) fungus Glomus intraradices. Examination of seedlings at weekly intewals indicated no fungal colonization until the third week when hyphae spread over the root surface. Following the development of appressoria, root penetration and intercellular colonization took place between the fourth and sixth weeks of inoculation. Mature fungal infections exhibiting vesicles became established after the sixth week. Key words: Parthenium argentaturn, VA mycorrhizas, Glomus intraradices. Vesicular-arbuscular mycorrhizal (VAM) associations are, at least in theory, of benefit to plants since they allow for an enhanced uptake of inorganic nutrients to the host when compared with non-mycorrhizal plants (Miller, Rajapakse & Garber, 1986). Reports also indicate that mycorrhizal plants may be more resistant to soilborne diseases and are possibly able to cope better under conditions of moisture stress (Dehne, 1982; Daniels-Hetrick, Gerschefske-Kitt & Thompson-Wilson, 1987). Guayule (Parthenium argenfafum Gray), a plant native to the Chihuahuan desert of northern Mexico and south western Texas, is well-suited to growth in arid, marginal areas (Hammond & Polhamus, 1965). In the U.S.A., native guayule is invaded and colonized by VAM fungi and a symbiont (Glomus intraradices Schenck & Smith) has been identified which greatly stimulates initial growth and eventual latex production (Bloss, 1980; Bloss & Pfeiffer, 1981, 1984). This study reports the temporal colonization by G. intradices of guayule seedlings grown in open pot culture, using light- and electron-microscope techniques.
Glomus intraradices inoculum consisting of spores, chopped roots and potting medium of Sudan grass (Sorghum vulgare var. sudanense (Piper) Hitchc.) grown in pot culture. The isolate was kindly provided by Dr H. E. Bloss. Seedlings were grown at a diurnal temperature ranging from 25 to 30 O C and were watered with full-strength Hoagland's nutrient solution with half-strength phosphate three times a week. Intermittent watering consisted of tap water. Seedlings were harvested weekly after emergence for a period of 8 wk. For comparison controls were grown without inoculum.
MATERIALS A N D METHODS
Root samples for scanning electron microscopy (SEM) were fixed in 3 % glutaraldehyde for 24 h before being dehydrated in a graded ethanol series ranging from 10 to TOO%and subsequently critical point dried. The dehydrated specimens were mounted on stubs and split longitudinally with a 'stickrip' method which involved lightly applying a piece of adhesive tape to the exposed surface of the mounted specimen. The tape was then ripped away so that the interior tissues under the epidermis were exposed. Stubs were then sputter
Inoculation technique
T o obtain optimum germination guayule seed was soaked in water for 4 h followed by a 2-h pre-treatment in a 200 mg 1-' gibberellin,,, solution, before being planted into seedling trays (50 an volume per cube) (Hurly, Van Staden & Smith, 1989). The growth medium used was acid-washed quartz sand (particle size 1 mm), which had been seeded with
Light microscopy
Roots (4 cm portions, cut to 1 cm lengths) to be subject to light microscopy (LM) were washed briefly before being fixed in formalin-acetic acid-ethanol-water (10 :5 :50 :35 v/v) overnight. They were then cleared and stained with chlorazol black E (Brundrett, Piche & Peterson, 1984). Slides were examined and photographed by bright field illumination. Scanning electron microscopy
Mycorrhizal colonization in guayule coated with gold palladium in a Polaron E5 100 vacuum evaporator, before examination by scanning electron microscopy. Transmission electron microscopy
Colonized root segments (1cm in length) were fixed in buffered 3 % glutaraldehyde for a minimum of 24 h (Lim, Fineran & Cole, 1983). For ease of handling all material was placed in plastic Eppendorf tubes for processing. Following post-fixation in 2 % osmium tetroxide, specimens were dehydrated in a graded ethanol series and embedded in Spurr's epoxy resin. Sections (0.05 pm) were cut on an ultramicrotome, collected on grids, and stained for 15 min each with aqueous 5 % uranyl acetate and lead citrate. Sections were viewed at 80 kV accelerating voltage.
RESULTS No colonization by the VAM fungus occurred until 3 wk after seedling emergence, when external hyphae had begun to spread over the root surface. Seedlings possessed many root hairs approximately 120 pm in length (Fig. I). No such colonization was observed in control guayule seedlings grown without inoculum in acid-washed sand in open pot culture. By the fourth week the fungus had penetrated the root through the development of appressoria (Fig. 2) which developed over the junction of two epidermal cells. Penetration was presumed to occur between the cells. Once in the root, hyphae became more septate, and this was followed by the rapid spread of intercellular hyphae throughout the root. Penetration of the root cells also occurred, and when this happened, hyphae became swollen and often completely occupied the interior of invaded epidermal cells. Scanning electron microscopy revealed that the swollen barrel-like hyphal cells were not completely separated from one another, but that connexions were maintained by perforations in the septum (Fig. 3). Intracellular hyphae contained dense cytoplasm (Fig. 4) and the plasma membrane was highly invaginated and appeared to be in a metabolically active state. An electron dense interfacial matrix completely surrounded the hyphae. Intrahyphal hyphae, in which the lumen of an outer, partly degenerate hypha appeared to contain an inner hypha with a denser, intact cytoplasm, were occasionally observed (Fig. 5). Hyphae in the middle and inner cortex characteristically produced coiled structures, while hyphal aggregates, which spread longitudinally between the cortical cells (Fig. 6) gave rise to arbuscules. The penetrating hyphae
832 formed the main arbuscule trunk and branched repeatedly (Fig. 7). These features, as well as degenerating arbuscules, were evident by the fifth week. By the sixth week vesicles were observed within the sites of colonization. They appeared as ovoid or elongated smoothwalled structures, which in most cases developed from intercalary hyphal swellings (Fig. 8). These structures later developed a thickened cell wall. Terminal swellings of the hyphae were also observed (Fig. 9). Crystalline structures occupying large areas of the cortical cell interior were often observed in mycorrhizal roots but were absent in non-mycorrhizal roots. Six wk after the guayule seedlings had germinated, Glornus infraradices had established all stages of the VAM symbiosis.
DISCUSSION These results closely correlate with those of Bloss & Pfeiffer (1984), with respect to the time required for mycorrhizal establishment. The initial two-week lag phase before colonization can be attributed to insufficient radicle area for effective fungal invasion. The results showed that after penetration of the root, the fungus rapidly colonized the outer cortical and sub-epidermal tissue layers. Arbuscule formation is rapid once the inner cortex has become colonized and their high metabolic activity accompanied by their rapid demise is indicated by the close proximity of young and degenerating arbuscules. In a comparative study of the anatomy of VAM fungi in subterranean clover, Abbott (1982) described the early stages of entry of three Glornus species. None of the species appears to develop similar pre-colonizing structures as G. intraradices. In addition to being swollen, these hyphae of G . intraradices contained higher numbers of septa. Septum formation is apparently not complete, but they possessed central pores. It may well be that this observation is typical for this particular host-mycobiont combination. Roots of Paspalurn notaturn colonized by G. intraradices have been shown to possess septa which are either non-perforate or contain fine perforations (Gibson, 1985). It is known that fungal hyphae produce septa in response to various deleterious physical or degenerative processes, such as wounding and arbuscule degeneration (Kinden & Brown, 1985). Thus the presence or absence of septa may be more a function of the physiological status of the VAM association than a diagnostic feature. Pores would allow for cytoplasmic continuity between the adjacent compartments and thus for the possible migration of the numerous nuclei which occur in intercellular hyphae (Bonfante-Fasolo, Berta & Fusconi, 1987). In addition, the
Figs 1-9. Colonization of payule roots by Glomw infraradices. Fig. I. Root surface colonization by fungal hyphae (H) between the root hairs (R) (Bar = 50 ym). Fig. 2. Appressorium formation on root epidermal cell (E) (Bar = 10 pm). Fig. 3. A single barrel-shaped hyphal segment showing a septum with a central pore (arrow) (Bar = 2 pm). Fig. 4. Transverse section through intracellular hypha showing vacuoles (V), endoplasmic reticulum (arrow), and root cell wall (W) (Bar = 0.5 wm). Fig. 5. Transverse section through an outer . 6. Development of hyphal strands within the intercellular hypha (OH) which encases an inner active hypha (IH) (Bar = 1 ~ m )Fig. spaces of the inner cortical cells (C) (Bar = 10 pm). Fig. 7. Young arbuscule showing hyphal branching beginning at the main arbuscule trunk (arrow) (Bar = 20 pm). Fig. 8. An elongated vesicle, with developed septa (arrow) (Bar = 20 pm). Fig. 9. Terminal swelling of a hypha with a single hyphal attachment (arrow) (Bar = 25 ym).
A. J. Vietti and J. van Staden
833
Mycorrhizal colonization in guayule translocation of polyphosphate-containing vacuoles from the external hyphae in the soil, t o the arbuscules in the cortical cells can possibly take place without much impediment. Glomtts intraradices, like a number of other Glornus species, has the ability t o initiate intrahyphal hyphae. The fungus relies o n this process after primary hyphae have degenerated or have become physically damaged. In this way the dead outer parent hyphae provide a path for new inner hyphae to reinfect previously infected areas after hyphal damage (Lim, Fineran & Cole, 1983). The appearance of crystals within the cells of mycorrhizal roots remains unexplained. They may be calcium carbonate or calcium oxalate deposits. Plants are known to deposit such crystals as a form of excretory product (Milne & Milne, 1959), but the role of these structures in mycorrhizal plants is unclear and deserves Further investigation.
Acknowledgements t o the Cooperative Scientific Programmes of the FRD for financial support and the Electron Microscope Unit in Pietermaritzburg for technical help. Dr Chris Walker is thanked for valuable comments and suggestions made o n this paper.
REFERENCES ABBOTT, L. K. (1982). Comparative study of vesicular-arbuscular mycorrhiza formed in subterranean clover. Australian Journal of Botany 30,485-499. BLOSS, H. E. (1980). Vesicular-arbuscular mycorrhiza in guayule (Parthenium argentatum). Mycologia 72, 213-216. BLOSS, H. E. & PFEIFFER, C. M. (1981). Growth and nutrition of mycorrhizal guayule plants. Annals of Applied Biology 99, 267-274. BLOSS, H. E. & PFEIFFER, C. M. (1984). Latex content and biomass
834 increase in mycorrhizal guayule (Parthenium argentatum)under field conditions. Annals of Applied Botany 104,175-183. BONFANTE-FASOLO, P., BERTA, G. & FUSCONI, A. (1987). Distribution of nuclei in a VAM fungus during its symbiotic phase. Transactions of the British Mycological Society 88, 263-266. BRUNDRETT, M. C., PICHE, Y. & PETERSON, R. L. (1984). A new method of observing the morphology of vesicular-arbuscular mycorrhizae. Canadian Journal of Botany 62, 2128-2134. DANIELS-HETRICK, B. A., GERSCHEFSKE-KITT, D. & THOMPSON-WILSON, G. (1987). Effect of drought stress on growth response in corn, Sudan grass and Big bluestem to Glomus etunicatum. New Phytologist 105,403-410. DEHNE, H. W. (1982). Interactions between vesicular-arbuscular mycorrhizal fungi and plant pathogens. Phytopafhology 72, 115-118. GIBSON, J. (1985). Morphology, cytology and ultrastructure of selected species of Endogonaceae (Endogonales: Zygomycetes). M.Sc. Thesis, University of Florida. HAMMOND, B. S. & POLHAMUS, L. F. (1965). Research on guayule (Parfhenium argentatum): 1942-1959. USDA Technical Bulletin 1327. HURLY, R. F., VAN STADEN, J. & SMITH, M. T. (1989). Guayule (Parfhenium argentatum Gray) seed germination. The effect of water soaks, sodium hypochlorite, gibberellic acid and gibberellin,,, applied as seed pre-treatments. Seed Science and Technology 17, 223-233. KINDEN, D. A. & BROWN, M. F. (1985). Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. 11. Intracellular hyphae and vesicles. Canadian Journal of Botany 21, 1768-1780. LIM, L. L., FINERAN, B. A. & COLE, A. L. J. (1983). Ultrastructure of intrahyphal hyphae of Glomus fasciculatum (Thaxter) Gerdemann and Trappe, in the roots of white clover (Trifolium repens L.). N m Phytologist 95, 231-239. MILLER, J. C., RAJAPAKSE, S. & GARBER, R. K. (1986). V-A mycorrhiza in vegetable crops. HortScience 21, 974-984. MILNE, L. J. & MILNE, M. (1959). Plant Life. Englewood Cliffs, NJ: Prentice-Hall.
(Received for publication 23 November 1988 and in revised form 6 November 1989)
Mycol. Res. 94 ( 6 ) :831-834 (1990) Printed in Great Britain
Histology of V-A mycorrhizal development in guayule seedlings
A. J. VIETTI A N D J. VAN STADEN UN/FRD Research Unit for Plant Growth and Development, Department of Botany, University of Natal, P 0 Box 375, Pietemritzburg 3200, Republic of South Africa
Histology of V-A mycorrhizal development in guayule seedlings. Mycological Research
94 ( 6 ) :831-834 (1990).
Guayule seed was germinated and grown in acid-washed quartz sand seeded with inoculum of the vesicular arbuscular mycorrhizal (VAM) fungus Glomus intraradices. Examination of seedlings at weekly intewals indicated no fungal colonization until the third week when hyphae spread over the root surface. Following the development of appressoria, root penetration and intercellular colonization took place between the fourth and sixth weeks of inoculation. Mature fungal infections exhibiting vesicles became established after the sixth week. Key words: Parthenium argentaturn, VA mycorrhizas, Glomus intraradices. Vesicular-arbuscular mycorrhizal (VAM) associations are, at least in theory, of benefit to plants since they allow for an enhanced uptake of inorganic nutrients to the host when compared with non-mycorrhizal plants (Miller, Rajapakse & Garber, 1986). Reports also indicate that mycorrhizal plants may be more resistant to soilborne diseases and are possibly able to cope better under conditions of moisture stress (Dehne, 1982; Daniels-Hetrick, Gerschefske-Kitt & Thompson-Wilson, 1987). Guayule (Parthenium argenfafum Gray), a plant native to the Chihuahuan desert of northern Mexico and south western Texas, is well-suited to growth in arid, marginal areas (Hammond & Polhamus, 1965). In the U.S.A., native guayule is invaded and colonized by VAM fungi and a symbiont (Glomus intraradices Schenck & Smith) has been identified which greatly stimulates initial growth and eventual latex production (Bloss, 1980; Bloss & Pfeiffer, 1981, 1984). This study reports the temporal colonization by G. intradices of guayule seedlings grown in open pot culture, using light- and electron-microscope techniques.
Glomus intraradices inoculum consisting of spores, chopped roots and potting medium of Sudan grass (Sorghum vulgare var. sudanense (Piper) Hitchc.) grown in pot culture. The isolate was kindly provided by Dr H. E. Bloss. Seedlings were grown at a diurnal temperature ranging from 25 to 30 O C and were watered with full-strength Hoagland's nutrient solution with half-strength phosphate three times a week. Intermittent watering consisted of tap water. Seedlings were harvested weekly after emergence for a period of 8 wk. For comparison controls were grown without inoculum.
MATERIALS A N D METHODS
Root samples for scanning electron microscopy (SEM) were fixed in 3 % glutaraldehyde for 24 h before being dehydrated in a graded ethanol series ranging from 10 to TOO%and subsequently critical point dried. The dehydrated specimens were mounted on stubs and split longitudinally with a 'stickrip' method which involved lightly applying a piece of adhesive tape to the exposed surface of the mounted specimen. The tape was then ripped away so that the interior tissues under the epidermis were exposed. Stubs were then sputter
Inoculation technique
T o obtain optimum germination guayule seed was soaked in water for 4 h followed by a 2-h pre-treatment in a 200 mg 1-' gibberellin,,, solution, before being planted into seedling trays (50 an volume per cube) (Hurly, Van Staden & Smith, 1989). The growth medium used was acid-washed quartz sand (particle size 1 mm), which had been seeded with
Light microscopy
Roots (4 cm portions, cut to 1 cm lengths) to be subject to light microscopy (LM) were washed briefly before being fixed in formalin-acetic acid-ethanol-water (10 :5 :50 :35 v/v) overnight. They were then cleared and stained with chlorazol black E (Brundrett, Piche & Peterson, 1984). Slides were examined and photographed by bright field illumination. Scanning electron microscopy
Mycorrhizal colonization in guayule coated with gold palladium in a Polaron E5 100 vacuum evaporator, before examination by scanning electron microscopy. Transmission electron microscopy
Colonized root segments (1cm in length) were fixed in buffered 3 % glutaraldehyde for a minimum of 24 h (Lim, Fineran & Cole, 1983). For ease of handling all material was placed in plastic Eppendorf tubes for processing. Following post-fixation in 2 % osmium tetroxide, specimens were dehydrated in a graded ethanol series and embedded in Spurr's epoxy resin. Sections (0.05 pm) were cut on an ultramicrotome, collected on grids, and stained for 15 min each with aqueous 5 % uranyl acetate and lead citrate. Sections were viewed at 80 kV accelerating voltage.
RESULTS No colonization by the VAM fungus occurred until 3 wk after seedling emergence, when external hyphae had begun to spread over the root surface. Seedlings possessed many root hairs approximately 120 pm in length (Fig. I). No such colonization was observed in control guayule seedlings grown without inoculum in acid-washed sand in open pot culture. By the fourth week the fungus had penetrated the root through the development of appressoria (Fig. 2) which developed over the junction of two epidermal cells. Penetration was presumed to occur between the cells. Once in the root, hyphae became more septate, and this was followed by the rapid spread of intercellular hyphae throughout the root. Penetration of the root cells also occurred, and when this happened, hyphae became swollen and often completely occupied the interior of invaded epidermal cells. Scanning electron microscopy revealed that the swollen barrel-like hyphal cells were not completely separated from one another, but that connexions were maintained by perforations in the septum (Fig. 3). Intracellular hyphae contained dense cytoplasm (Fig. 4) and the plasma membrane was highly invaginated and appeared to be in a metabolically active state. An electron dense interfacial matrix completely surrounded the hyphae. Intrahyphal hyphae, in which the lumen of an outer, partly degenerate hypha appeared to contain an inner hypha with a denser, intact cytoplasm, were occasionally observed (Fig. 5). Hyphae in the middle and inner cortex characteristically produced coiled structures, while hyphal aggregates, which spread longitudinally between the cortical cells (Fig. 6) gave rise to arbuscules. The penetrating hyphae
832 formed the main arbuscule trunk and branched repeatedly (Fig. 7). These features, as well as degenerating arbuscules, were evident by the fifth week. By the sixth week vesicles were observed within the sites of colonization. They appeared as ovoid or elongated smoothwalled structures, which in most cases developed from intercalary hyphal swellings (Fig. 8). These structures later developed a thickened cell wall. Terminal swellings of the hyphae were also observed (Fig. 9). Crystalline structures occupying large areas of the cortical cell interior were often observed in mycorrhizal roots but were absent in non-mycorrhizal roots. Six wk after the guayule seedlings had germinated, Glornus infraradices had established all stages of the VAM symbiosis.
DISCUSSION These results closely correlate with those of Bloss & Pfeiffer (1984), with respect to the time required for mycorrhizal establishment. The initial two-week lag phase before colonization can be attributed to insufficient radicle area for effective fungal invasion. The results showed that after penetration of the root, the fungus rapidly colonized the outer cortical and sub-epidermal tissue layers. Arbuscule formation is rapid once the inner cortex has become colonized and their high metabolic activity accompanied by their rapid demise is indicated by the close proximity of young and degenerating arbuscules. In a comparative study of the anatomy of VAM fungi in subterranean clover, Abbott (1982) described the early stages of entry of three Glornus species. None of the species appears to develop similar pre-colonizing structures as G. intraradices. In addition to being swollen, these hyphae of G . intraradices contained higher numbers of septa. Septum formation is apparently not complete, but they possessed central pores. It may well be that this observation is typical for this particular host-mycobiont combination. Roots of Paspalurn notaturn colonized by G. intraradices have been shown to possess septa which are either non-perforate or contain fine perforations (Gibson, 1985). It is known that fungal hyphae produce septa in response to various deleterious physical or degenerative processes, such as wounding and arbuscule degeneration (Kinden & Brown, 1985). Thus the presence or absence of septa may be more a function of the physiological status of the VAM association than a diagnostic feature. Pores would allow for cytoplasmic continuity between the adjacent compartments and thus for the possible migration of the numerous nuclei which occur in intercellular hyphae (Bonfante-Fasolo, Berta & Fusconi, 1987). In addition, the
Figs 1-9. Colonization of payule roots by Glomw infraradices. Fig. I. Root surface colonization by fungal hyphae (H) between the root hairs (R) (Bar = 50 ym). Fig. 2. Appressorium formation on root epidermal cell (E) (Bar = 10 pm). Fig. 3. A single barrel-shaped hyphal segment showing a septum with a central pore (arrow) (Bar = 2 pm). Fig. 4. Transverse section through intracellular hypha showing vacuoles (V), endoplasmic reticulum (arrow), and root cell wall (W) (Bar = 0.5 wm). Fig. 5. Transverse section through an outer . 6. Development of hyphal strands within the intercellular hypha (OH) which encases an inner active hypha (IH) (Bar = 1 ~ m )Fig. spaces of the inner cortical cells (C) (Bar = 10 pm). Fig. 7. Young arbuscule showing hyphal branching beginning at the main arbuscule trunk (arrow) (Bar = 20 pm). Fig. 8. An elongated vesicle, with developed septa (arrow) (Bar = 20 pm). Fig. 9. Terminal swelling of a hypha with a single hyphal attachment (arrow) (Bar = 25 ym).
A. J. Vietti and J. van Staden
833
Mycorrhizal colonization in guayule translocation of polyphosphate-containing vacuoles from the external hyphae in the soil, t o the arbuscules in the cortical cells can possibly take place without much impediment. Glomtts intraradices, like a number of other Glornus species, has the ability t o initiate intrahyphal hyphae. The fungus relies o n this process after primary hyphae have degenerated or have become physically damaged. In this way the dead outer parent hyphae provide a path for new inner hyphae to reinfect previously infected areas after hyphal damage (Lim, Fineran & Cole, 1983). The appearance of crystals within the cells of mycorrhizal roots remains unexplained. They may be calcium carbonate or calcium oxalate deposits. Plants are known to deposit such crystals as a form of excretory product (Milne & Milne, 1959), but the role of these structures in mycorrhizal plants is unclear and deserves Further investigation.
Acknowledgements t o the Cooperative Scientific Programmes of the FRD for financial support and the Electron Microscope Unit in Pietermaritzburg for technical help. Dr Chris Walker is thanked for valuable comments and suggestions made o n this paper.
REFERENCES ABBOTT, L. K. (1982). Comparative study of vesicular-arbuscular mycorrhiza formed in subterranean clover. Australian Journal of Botany 30,485-499. BLOSS, H. E. (1980). Vesicular-arbuscular mycorrhiza in guayule (Parthenium argentatum). Mycologia 72, 213-216. BLOSS, H. E. & PFEIFFER, C. M. (1981). Growth and nutrition of mycorrhizal guayule plants. Annals of Applied Biology 99, 267-274. BLOSS, H. E. & PFEIFFER, C. M. (1984). Latex content and biomass
834 increase in mycorrhizal guayule (Parthenium argentatum)under field conditions. Annals of Applied Botany 104,175-183. BONFANTE-FASOLO, P., BERTA, G. & FUSCONI, A. (1987). Distribution of nuclei in a VAM fungus during its symbiotic phase. Transactions of the British Mycological Society 88, 263-266. BRUNDRETT, M. C., PICHE, Y. & PETERSON, R. L. (1984). A new method of observing the morphology of vesicular-arbuscular mycorrhizae. Canadian Journal of Botany 62, 2128-2134. DANIELS-HETRICK, B. A., GERSCHEFSKE-KITT, D. & THOMPSON-WILSON, G. (1987). Effect of drought stress on growth response in corn, Sudan grass and Big bluestem to Glomus etunicatum. New Phytologist 105,403-410. DEHNE, H. W. (1982). Interactions between vesicular-arbuscular mycorrhizal fungi and plant pathogens. Phytopafhology 72, 115-118. GIBSON, J. (1985). Morphology, cytology and ultrastructure of selected species of Endogonaceae (Endogonales: Zygomycetes). M.Sc. Thesis, University of Florida. HAMMOND, B. S. & POLHAMUS, L. F. (1965). Research on guayule (Parfhenium argentatum): 1942-1959. USDA Technical Bulletin 1327. HURLY, R. F., VAN STADEN, J. & SMITH, M. T. (1989). Guayule (Parfhenium argentatum Gray) seed germination. The effect of water soaks, sodium hypochlorite, gibberellic acid and gibberellin,,, applied as seed pre-treatments. Seed Science and Technology 17, 223-233. KINDEN, D. A. & BROWN, M. F. (1985). Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. 11. Intracellular hyphae and vesicles. Canadian Journal of Botany 21, 1768-1780. LIM, L. L., FINERAN, B. A. & COLE, A. L. J. (1983). Ultrastructure of intrahyphal hyphae of Glomus fasciculatum (Thaxter) Gerdemann and Trappe, in the roots of white clover (Trifolium repens L.). N m Phytologist 95, 231-239. MILLER, J. C., RAJAPAKSE, S. & GARBER, R. K. (1986). V-A mycorrhiza in vegetable crops. HortScience 21, 974-984. MILNE, L. J. & MILNE, M. (1959). Plant Life. Englewood Cliffs, NJ: Prentice-Hall.
(Received for publication 23 November 1988 and in revised form 6 November 1989)