Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2000-12-15
2003-06-10
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S254100, C047S001100, C071S005000
Reexamination Certificate
active
06576457
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to novel fungal growth media and methods for the in vitro aseptic mass production of endomycorrhizal fungal propagules, mainly spores, using transformed root organ culture.
BACKGROUND OF THE INVENTION
Vesicular-arbuscular mycorrhizal (VAM) fungi form beneficial symbiotic associations with roots of a wide range of plants (Harley and Smith, 1983). The term “mycorrhiza,” which literally means “fungus root,” was first used in 1885 by A. B. Frank to describe the intimate associations between fungal hyphae and the roots of forest trees. The hyphae of VA mycorrhizal fungi penetrate and form arbuscules within root cortical cells and intercellular vesicles, but the external hyphae extend further into the soil for mineral nutrient uptake. A simple interpretation of mycorrhizal symbiosis is that the plant supplies carbon compounds to increase fungal growth and the plant benefits by having its root system effectively extended. The surface area of fungal hyphae can be much greater than that of the plant roots (Smith and Gianinazzi-Pearson; 1992, Smith and Read, 1997).
The importance of VA mycorrhizae in the growth and nutrition of plants has recently been reviewed (Gianinazzi and Schhepp, 1994; Smith and Read, 1997; Harrison, 1999). On soils low in available phosphate, the improved growth observed in VA mycorrhizal plants compared with uninoculated controls is thought to be due largely to an improved supply of phosphate. The external hyphae in soil absorbs non-mobile nutrients such as phosphate (P), potassium (K
+
), and ammonium (NH
4
+
) beyond the root zone and transfer phosphate from the fungus to the root cells. Thus, any process that was previously limited by the availability of phosphate will increase in rate. Improved phosphate nutrition, as well as direct fungal effects, may be implicated in the enhanced uptake of other macronutrients and micronutrients by plants.
Many VA mycorrhizal plants have increased resistance to disease compared to non-mycorrhizal controls, although the converse has also been reported. It has been suggested that physical protection of potential entry points on the root surface by the mycorrhizal fungi, and the reduced availability of carbon compounds to pests and pathogens (due to their being used by the fungi) are responsible for increased resistance to some diseases; however, the improved nutritional status of mycorrhizal plants made them more susceptible to other diseases (Pfleger and Linderman, 1994; Podila and Dodds,1999). A positive role for VA mycorrhizae in plant protection is indicated by the presence of phytoalexins (compounds involved in resistance to infection by pathogens), for example in mycorrhizal soybean roots (Morandi et al., 1984; Morandi, 1989; Harrison, 1999).
Certain changes in the plant hormonal balance have been shown to be related to a VA mycorrhizal effect. Some VA endomycorrhizae have been shown to contain elevated levels of phytohormones compared with non-infected roots. This may be due to the improved supply of nutrients, but could equally well be a direct result of the fungus, as it has been reported that plant growth promoting substances have been detected in germinating VA mycorrhizal fungal spores. Hormone accumulation in host tissue is affected by mycorrhizal infection, with changes in the levels of cytokinin, abscisic acid, and gibberellin-like substances and alteration in biomass partitioning (Barker et al., 1998; Danneberg et al., 1992; Dixon, 1992; Goicoechea, 1996).
The effect of VAM fungal infection on the drought resistance of plants is probably also due to the improvement of nutrient uptake. A mycorrhizal pathway of nutrient acquisition would become much more important in dry soil, because the nutrients become much less mobile. There seems little doubt that, like plant growth changes, mycorrhizal fungi can cause changes in plant water relations and may, at least in some cases, improve drought resistance of the plant (Al-Karaki et al., 1998; Goicoevhea et al., 1996; Nelson, 1987).
Morton and Benny (1990) described 149 species of VAM fungi. The VA mycorrhizal fungi are all classified as belonging to the family Endogonaceae possessing coenocytic hyphae (with only rare septa or none at all), which contain many nuclei. They have not propagated independently from host plant roots, and no sexual reproduction has observed, although both have been demonstrated in some non-mycorrhizal, saprophytic members of the Endogonaceae. Genera known to form VA mycorrhizae are Glomus, Gigaspora, Acaulospora, Sclerocystis, and Entrophospora. Knowledge of the phylogenetic relationships of VAM fungi is fragmentary. There is scant literature available on cytological or sexual processes involved in spore formation for these fungi. The taxonomic position of the VAM fungi is, therefore, inferred from developmental processes and spore morphology (Schenck and Yvonne, 1992; Morton, 1995).
The VA mycorrhizal fungi produce large vegetative spores (often greater than 100 &mgr;m in diameter) usually on hyphae external to the roots. The genera have been classified according to the appearance of the spores. Spores may be recovered from the soil around infected plants by wet-sieving through a series sieves of different sizes, ranging from 1,000 &mgr;m to 75 &mgr;m. Either spores or fragments of infected roots may be used to inoculate further plants. Spores produced by Gigaspora, Acaulospora, and Entrophospora are called azygospores. Spores produced by Glomus and Sclerocystis, termed chlamydospores, are presumably asexual, thick-walled resting cells. Additional morphological characters that are useful in separating genera and species of VAM fungi are (i) the method of spore germination; (ii) presence or absence of sporocarps; (iii) presence or absence of auxiliary cells; (iv) spore dimensions; (v) spore color; (vi) spore ornamentation; (vii) number and type of spore walls; and (viii) histochemical reactions (Hall, 1984; Morton, 1988, 1995; Schenck and Yvonne, 1992).
Spores of several species of VAM fungi readily germinate on distilled water or semi-solid water agar, suggesting that nutritional requirements for germination are met by the mobilization of spore reserves. In some instances, an exogenous supply of nutrients may result in increased germination rates; however, no specific requirement for germination on water agar has been reported. The germination rates of spores of VAM fungi are improved by thiamine, nutrient broth medium, and soil extracts, root exudates, soil volatile compounds and small amounts of glucose (Carr et al., 1985; Carr et al., 1986; Elias and Safir, 1987; Graham, 1982; Hepper, 1979, 1983, 1984; Mosse, 1959; Smith and Gianinazzi-Pearson, 1988). Some early biochemical events in the germination of VAM fungal spores have been studied. A number of enzyme activities have been demonstrated in germinating spores: glutamate dehydrogenase suggesting amino acid respiration; succinate dehydrogenase suggesting the Krebs (TCA) cycle; glyceraldehyde-3-phosphate dehydrogenase indicating the Embden-Myerhof-Parnas glycolytic pathway; and glucose 6-phosphate dehydrogenase, which suggests the presence of the hexose monophosphate shunt. However, it is necessary to demonstrate all the relevant enzymes of these pathways to confirm their full metabolic functions (Beilby, 1982, 1983; Macdonald and Lewis, 1978). Acetate is incorporated into organic and amino acids, indicating that the TCA cycle and amino acid biosynthetic pathways are operative. Nuclear DNA synthesis has not been detected during germination, but nuclear division does occur (Burggraffand Beringer, 1989; Smith and Gianinazzi-Pearson, 1988).
In summary, spores of VAM fungi seem to be able to germinate readily, but not continuous growth and sporulation unless the fungal hyphae form symbiotic relationship with living roots. During root-fungus interactions, signal molecules from host plants are probably produced to turn on the fungal genes for growth and development. The symbiotic biology involved should be a focus of study in order to desi
Connor Margaret A.
Fado John
Marx Irene
The United States of America as represented by the Secretary of
Wai Thanda
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