Sporogenesis-EKTN

Spore development of Entrophospora kentinensis in an aeroponic system

Chi-Guang Wu, Yen-Sher Liu

Soil Microbiology Lab, Agricultural Chemistry Department, Taiwan Agricultural Research Institute, Wu-feng, Taichung, Taiwan, Republic of China.

Ling-Ling Hung

US Public Health Service, Division of Federal Occupational Health, Philadelphia, Pennsylvania

Abstract: Entrophospora kentinensis was propagated with bahia grass and sweet potato in an aeroponic system. Spores were produced six weeks later after host plants were transferred to an aeroponic chamber. Sporiferous saccules were formed either by external mycelium or within roots. Sporogenesis started inside the stalk of a sporiferous saccule by the formation of a septum in the lower channel of the stalk. The cytoplasm within the saccule seemed the major source for the spore formation. When the cytoplasm flowed downward, the central part of stalk swelled and tiny vacuoles were formed at the periphery of saccule. As the primordial spore was differentiated inside the stalk, another septum was formed right below the saccule. After spores mature, the terminal vesicles of saccules and the lower hyphal stalks degenerate and leave two scars. Differences in spore ontogeny between Acaulospora and Entrophospora are discussed.

Key Words: Acaulospora, aeroponic culture, Entrophospora, sporogenesis.

INTRODUCTION

Entrophospora kentinensis Wu & Liu is a newly described species which was isolated originally from the rhizosphere of native weeds (Setaria viridis (L.) Beauv.) in National Kentin Park, Southern Taiwan (Wu, et al., 1995). The species occurs throughout Taiwan in soil of a pH range from 5.71 to 7.94. In a fallow field heavily polluted by copper waste up to 233 ppm, the hyphae of E. kentinensis effectively colonized the roots of Eleusine indica (L.) Goertn., Paspalum spp. and other grasses.

Up to now, three major methods have been commonly used for inoculum production of arbuscular mycorrhizal fungi (AMF)-(i) pot culture, (ii) solution culture, and (iii) root organ culture. Traditionally, AMF are cultivated by sand-based pot cultures (Menge, 1984). However, it is a labor-intensive process and not economical for mass production of inoculum. Solution culture techniques such as nutrient film (Elms and Mosse, 1984; Mosse and Thompson, 1984) and aeroponics (Sylvia and Hubbell, 1986) are mainly adapted for the production of clean, soilless inoculum of AMF. Nevertheless, these methods have proven successful only in cultivating Glomus species (Hung and Sylvia, 1988; Sylvia and Jarstfer, 1994). Recently, several tissue culture techniques for producing contaminant-free AMF inoculum have succeeded. Ri-plasmid transformed-root cultures seem to offer the most efficient method for growing colonized roots (Mugnier and Mosse, 1987). Piche and his colleagues (Becard and Piche, 1989;1990; Chabot, et al.,1992a; 1992b) completed excellent studies on the root organ cultures of Glomus intraradices Schenck & Smith and Gigaspora margarita Becker & Hall.

In this study, we report cultivation of Entrophospora kentinensis in an aeroponic chamber and describe spore ontogeny.

MATERIALS AND METHODS

The spores of Entrophospora kentinensis (SM-22) were initially isolated from field soil by wet-sieving and decanting (Gerdemann and Nicolson, 1963). The sievings were further purified by sucrose centrifugation (Jenkins, 1964). Spores were then propagated in sand-based pot cultures in the green house with bahia grass (Paspalum notatum Flü gge) and sweet potato (Ipomoea batatas (L.) Lam. var. edulis Mak.). In the study of spore development in aeroponic culture, spores were mounted in PVLG (polyvinyl alcohol-lactic acid-glycerol) (Koske and Tessier, 1983) or Hoyer's medium and made into semipermanent slides. Microscopic observations were made and photographed with differential interference contrast and phase contrast microscopes. Some collections were also preserved in FAA fixative.

Aeroponic cultures were started with precolonized seedlings of bahia grass and sweet potato. Surface-disinfected seeds of bahia grass and slips of sweet potato were placed in the soil inoculum of E. kentinensis in shallow trays. The inoculum was increased with bahia grass and the culture medium contained sand and hydrocorn (10:1). Seedlings were grown in the greenhouse for 2-3 weeks. Root colonization by AMF was confirmed by root staining (Phillips and Hayman, 1970). Colonized seedlings were placed into aeroponic chambers in a nonshaded greenhouse with mean max. and min. temperatures of 22.8 and 34.70C and mean max. radiation of 127.34 MJm-2day-1. The apparatus of aeroponic chambers approximately 61 cm W x 122 cm L x 61 cm D, was adapted from Hung and Sylvia (1988) and Zobel, et al.(1976). Dilute Hoagland's nutrient solution (about 70 liter) was used in the aeroponic chamber, with the solution pH initially adjusted to 6.5 with 1N NaOH (Sylvia and Hubbell, 1986). The root segments were regularly checked with a dissecting microscope every two weeks for sporulation and then cleared in 10% KOH and stained with 0.05% trypan blue.

The spore ontogeny of E. kentinensis was compared with Acaulospora scrobiculata Trappe (1977). The spores of A. scrobiculata (SM-18) were maintained in sandy pot cultures.

RESULTS AND DISCUSSION

Entrophospora kentinensis typically produces spores singly in soil or in roots and forms typical vesiclular-arbuscular mycorrhizae. In this study, the spores were produced six weeks after host plants were transferred to an aeroponic chamber. Various stages of spore ontogeny could be found within root tissue or in external mycelium. External mycelium is 2.5-3 (-3.5) mm diam, dichotomously branched, and is similar to that of other AMF.

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Spore formation is initiated by a slightly tapering hypha, terminating in a globose to subglobose swollen saccule, 100-150 mm diam (FIG. 1). The saccule is white to subhyaline and filled with dense cytoplasm. Before spore formation, a septum appears in the lower part of the hyphal stalk of the saccule and other delicate branches arise from the stalk (FIGS. 1, 2). The cytoplasm within the saccule appears to flow toward the septum and increases turgor pressure in the stalk (FIG. 3), accompanyed by the formation of peripheral vacuoles inside the saccule (FIG. 4). While the cytoplasmic stream reaches into the stalk, a finger-like structure of the plasm enclosed by a membranous wall extends toward the septum (FIG. 2). As a certain amount of cytoplasm is squeezed into the stalk, a primordial spore is differentiated inside and at the same time the other septum laid at the upper part of the stalk segregates the spore from the saccule (FIGS. 5-6). The pitted ornamentation on the spore is visible at this stage (FIGS. 6-7). Spores at first are white to subhyaline and become pale yellow to yellow brown at maturity, globose, subglobose, ellipsoidal, irregular, 85-140 x 95-210 mm. At the final stage, the residual cytoplasm inside the saccule lyses and the contents of saccule become transparent (FIG. 8). As the spore matures, the saccule and hyphal stalk degnerate and leave two scars on the surface (FIG. 8). One is larger, 20-32.5 mm diam, at the position of setum between spore and saccule, and the other is smaller, 5-6 mm diam, at the position of septum separating the spore from the hyphal stalk. The latter was visible only on the newly-formed spore.

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In this study, spore wall formation begins as early as when the cytoplasm streams downward toward the septum in the lower part of hyphal stalk. The tip of cytoplasmic front appears obviously coated by a layer of wall material (FIGS. 5-7) and before the second septum is formed, the process of wall structure differentiation was already initiated. An ultrastructural study of the details of wall formation will be continued.

Fig. 9. Mature spore of E. kentinensis. Note the inner amorphous layer.

Fig. 10. Wall structure of E. kentinensis. Spore surface ornamented with pits.

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Sporogenesis of Entrophospora is different from that of Acaulospora (Ames and Schneider, 1979). Spores of Acaulospora are produced laterally on the stalk of a large, terminal thin-walled saccule. Similar to that found in Entrophospora, there is also a septum formed in the lower part of hyphal stalk at the early stage of sporogenesis. Because of the occlusion by the septum, the turgor pressure will be intensified as the cytoplasm is squeezed into the hyphal stalk due to the formation of peripheral vesicles in the saccule. Consequently, in the case of Acaulospora, there appears to be an enzymatic softening of an area on the lateral wall of the stalk. At this point the turgor pressure distends the stalk on one side where it swells up to form a globular vesicle in which a spore is produced. Based on an observation that a mature spore of Acaulospora is very often attached by a circular neck or scar, the presumed reaction site of enzyme activity on the lateral side of stalk is around 10-15 mm diam. InEntrophospora, however, the enzymatic reaction area for the expansion of the hyphal stalk to accomodate the developing spore includes a larger portion of the stalk in the area between the two septa.

ACKNOWLEDGMENTS

This research was financially supported by the National Science Council (NSC 83-0409-B-055-008) and the Council of Agriculture (83-AST-FAD2,6-57), Exective Yuan, Taiwan, Republic of China. Appreciation is extended to Drs. R. E. Koske, C. Walker, J. W. Kimbrough, and G. L. Benny for their critical reviews of this manuscript.

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