Using Flavonoids to Control in vitro Development of Vesicular Arbuscular Mycorrhizal Fungi

Abstract

Vesicular-arbuscular mycorrhizal (VAM) fungi and other beneficial rhizosphere microorganisms, such as Rhizobium bacteria, must locate and infect a host plant before either symbiont profits. Although benefits of the VAM association for increased phosphorous uptake have been widely documented, attempts to improve the fungus and to produce agronomically useful amounts of inoculum have failed due to a lack of in vitro production methods. This project was designed to extend our prior observation that the alfalfa flavonoid quercetin promoted spore germination and hyphal growth of VAM fungi in the absence of a host plant. On the Israeli side of the project, a detailed examination of changes in flavonoids and flavonoid-biosynthetic enzymes during the early stages of VAM development in alfalfa found that VAM fungi elicited and then suppressed transcription of a plant gene coding for chalcone isomerase, which normally is associated with pathogenic infections. US workers collaborated in the identification of flavonoid compounds that appeared during VAM development. On the US side, an in vitro system for testing the effects of plant compounds on fungal spore germination and hyphal growth was developed for use, and intensive analyses of natural products released from alfalfa seedlings grown in the presence and absence of microorganisms were conducted. Two betaines, trigonelline and stachydrine, were identified as being released from alfalfa seeds in much higher concentrations than flavonoids, and these compounds functioned as transcriptional signals to another alfalfa microsymbiont, Rhizobium meliloti. However, these betaines had no effect on VAM spore germination or hyphal growth i vitro. Experiments showed that symbiotic bacteria elicited exudation of the isoflavonoids medicarpin and coumestrol from legume roots, but neither compound promoted growth or germination of VAM fungi in vitro. Attempts to look directly in alfalfa rhizosphere soil for microbiologically active plant products measured a gradient of nod-gene-inducing activity in R. meliloti, but no novel compounds were identified for testing in the VAM fungal system in vitro. Israeli field experiments on agricultural applications of VAM were very successful and developed methods for using VAM to overcome stunting in peanuts and garlic grown in Israel. In addition, deleterious effects of soil solarization on growth of onion, carrot and wheat were linked to effects on VAM fungi. A collaborative combination of basic and applied approaches toward enhancing the agronomic benefits of VAM asociations produced new knowledge on symbiotic biology and successful methods for using VAM inocula under field conditions