1.1.1. L793...’ 3.. .1) . .i. . .I... .5... .2. if . . . a)! . p .. i233? . Q. Y. \ 3.4(1‘:.§JM..$I¢Lc .4: ..v . . \r. 4: . u.- .ux.ll~‘.l .? IN" 15... .3. . ishct~ ‘1: I: 4. Aux‘r.) .. ‘7‘: Bikz I» r020 .v‘ it"s: «I \ u. a... .1. I o Fvaaizu? . lillllll‘lillillll LIBRARY Michigan state University THESIS This is to certify that the dissertation entitled Physiological Alterations in Mycorrhizal Plants as Affected by Phenolic Compounds presented by Leadir Lucy Martins Fries has been accepted towards fulfillment of the requirements for Ph.D. degree in Botany & Plant Pathology fixfiJgg Major professor Date (fit/9.5.. MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 PLACE ll RETURN BOX to romovo this chookout horn your rooord. TO AVOID FINES return on or odor. dot. duo. DATE DUE DATE DUE DATE DUE MSU In An Nflmotivo Mon/Emu Opportunity Institqun W PHYSIOLOGICAL ALTERATIONS IN MYCORRHIZAL PLANTS AS AFFECTED BY PHENOLIC COMPOUNDS By Leadir Lucy Martins Fries A DISSERTATION Submitted to Michigan State University in partial fulfillment of requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1 995 ABSTRACT PHYSIOLOGICAL ALTERATIONS IN MYCORRHIZAL PLANTS AS AFFECTED av PHENOLIC COMPOUNDS By Leadir L. M. Fries Early events in the colonization Of vesicular-arbuscular myccrrhizal (VAM) fungi on host roots were monitored by analyzing selected isoenzyme markers. The rates and amount Of colonization were adjusted using different concentrations Of phenolics and inorganic phosphorus. Certain isoenzymes Of malate dehydrogenase, acid and alkaline phosphatase, esterase and peroxidase were affected by either the rate Of VAM fungal spread or the total fungal biomass found in corn rOOts. Specific isozymes were identified and were useful tO monitor the physiology Of the initial events in VAM symbioses. Forrnononetin, an isoflavone, increased VAM colonization at all phosphorus (P)-levels indicating that it is useful in overcoming P inhibition Of VAM colonization. Peroxidase activity, which increases with soil available P, was decreased following the application Of formononetin in both VAM and non-VAM roots, shown here for the first time. It was also found that certain phenolic compounds, such as quercetin, can stimulate or inhibit VAM fungal infection depending on the concentration applied to the soil. Several phenolics commonly found in the Graminae, such as p-coumaric acid, had a dramatic effect on sorghum growth and rOOt colonization. The performance Of endomycorrhizal symbiosis in the field can potentially be affected by the presence Of these compounds, which are commonly exuded by plants. Specific isozyme activities were correlated with fungal biomass, fungal spread, host-plant response tO the fungus, and general metabolic activity. Due tO fcrrnononetin’s inhibition Of peroxidase activity, this compound may enable mycorrhizal fungi to spread through the host rOOt with greater ease since there are less cell wall crosslinks. For effective management and utilization Of mycorrhizal fungi in the field, it is suggested that both phenolic and P status should be monitored prior tO application Of P fertilizers or iSOflavoneS. iv In memory Of my father LeOdonIO who’s inspiration has always been present. ACKNOWLEDGMENTS I am grateful tO all people who has profoundly affected my scientific development, in particular for the support and insights Of Dr. Gene Safir, my major professor, and Dr. Raymond Hammerschmidt, Dr. Muraleedharan Nair, and Dr. Alvin Smucker, members Of my advisory commitee. Additionally, I owe much gratitude tO Raymond Pacovsky for great discussions and friendship. I would also like to thank Dr. Jose Siqueira, Dr. Dalvan Reinert, and Dr. Joao Kaminski for their encouragement in pursuing my doctoral degree. TO the Brazilian government through MEC-CAPES for the scholarship and Michigan State University, especially Botany and Plant Pathology Department, for accepting me in its Graduate Program. TO my lab COllegues and friends, Aysegul, Sarah, Jacqueline, Eliana, Brendan, Richard, Adnan, and Virginia, who will always be in my thoughts for the moments we shared. TO my Brazilian friends Angela, Carlos, Rita, Ivan, Fatima, Henrique, lsabela, Jose Maria, Anette, Ricardo and Clarice for their loving support. From the bottom Of my heart, I am specially thankful tO my mom, Adiles, my husband Marcos, and my children, Milton, Kalynka and Henrique who always had encouraging, understanding and loving words and whose struggles and battles are the same as mine. vi TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................... ix LIST OF FIGURES ..................................................................................................................... x CHAPTER 1 ................................................................................................................................ 1 INTRODUCTION ..................................................................................................................... 1 Mycorrhizae ............................................................................................................................. 1 Germination process ................................................................................................................ 2 Establishment Of mycorrhizae .................................................................................................. 3 Plant reactions to VAM fungi .................................................................................................... 5 Plant growth responses to VAM symbiosis ............................................................................... 6 Applications of isozymes in VAM research .............................................................................. 8 Role Of phenolics in VAM symbioses ...................................................................................... 11 List Of references ................................................................................................................... 15 CHAPTER 2 .............................................................................................................................. 26 EXPRESSION OF ISOENZY MES ALTERED BY BOTH Glomus intratadices COLONIZATION AND FORMONONETIN APPLICATION IN CORN (Zea mays L.) ROOTS ................................................................................................................................. 26 Introduction .............. ’ .............................................................................................................. 2 6 Material and Methods ............................................................................................................ 28 Experimental design ........................................................................................................... 28 Growth conditions ............................................................................................................... 29 Evaluations and assays ...................................................................................................... 30 Preparation Of root extracts ................................................................................................ 30 Polyacrylamide native gel electrophoresis ........................................................................... 31 Total peroxidase activity measurements ............................................................................. 31 Statistical analysis .............................................................................................................. 32 Results .................................................................................................................................. 32 Growth and colonization ..................................................................................................... 32 MAD-malate dehydrogenase ............................................................................................... 34 Esterase ............................................................................................................................. 34 Peroxidase isozymes .......................................................................................................... 39 Total peroxidase ................................................................................................................. 39 Discussion ............................................................................................................................. 41 List Of references ................................................................................................................... 45 vii CHAPTER 3 ............................................................................................................................. 50 PHOSPHORUS EFFECT ON ISOENZYME EXPRESSION IN ENDOMYCORRHIZAL CORN .................................................................................................................................... 50 Introduction ............................................................................................................................ 50 Material and Methods ............................................................................................................ 54 Experimental design ........................................................................................................... 54 Growth conditions ............................................................................................................... 55 Growth response and colonization ...................................................................................... 56 Preparation Of root extracts ................................................................................................ 57 Polyacrylamide native gel electrophoresis ........................................................................... 57 Total acid and alkaline phosphatase measurements ........................................................... 57 Total peroxidase activity measurements ............................................................................. 58 Statistical analysis .............................................................................................................. 59 Results .................................................................................................................................. 59 Growth ............................................................................................................................... 59 Colonization ....................................................................................................................... 61 Total acid phosphatase activity ........................................................................................... 61 Total alkaline phosphatase activity ..................................................................................... 64 Acid Phosphatase isozymes ............................................................................................... 64 NAB-malate dehydrogenase isozymes ............................................................................... 68 Esterase isozymes ............................................................................................................. 68 Total peroxidase activity ..................................................................................................... 68 Peroxidase isozymes .......................................................................................................... 76 Discussion ............................................................................................................................. 76 List Of references ................................................................................................................... 86 CHAPTER 4 .............................................................................................................................. 94 PLANT GROWTH AND ARBUSCULAR MYCORRHIZAL (Glomus intramdices) COLONIZATION AFFECTED BY EXOGENOUSLY APPLIED PHENOLIC COMPOUNDS 94 Introduction ............................................................................................................................ 94 Material and Methods ............................................................................................................ 96 Plant, soil and fungal material ............................................................................................. 96 Phenolic compounds .......................................................................................................... 97 Experiments ....................................................................................................................... 97 Growth conditions ............................................................................................................. 100 Experimental design and statistical analysis ..................................................................... 100 Results ................................................................................................................................ 101 Discussion ........................................................................................................................... 1 15 List of references ................................................................................................................. 124 CHAPTER 5 ............................................................................................................................ 131 Summary and conclusions ....................................................................................................... 131 viii Table Table 2.1. Table 3.1. LIST OF TABLES Page Plant growth and root colonization of corn plants at 1, 2 and 3 weeks Of growth following Glomus intraradices (+VAM) inoculation or fcrrnononetin (+FOR) application. Plants were harvested at 1, 2, and 3 weeks of growth ................................................................................................................ 33 Growth, root phosphorus and root protein concentration Of corn plants inoculated with Glomus intraradices (+VAM) and fcrrnononetin (+FOR) as affected by different levels Of phosphorus applied in the soil. Plants were harvested at 3 weeks Of growth ................................................................................................................ 60 LIST OF FIGURES Figure Page Figure 2.1. Banding pattern for MAD-malate dehydrogenase (MDH) isoenzymes visualized in a 7.5 % polyacrylamide native gel. Protein extracts (30 mg/Iane) from one, two, and three—week-Old Zea mays roots. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and '+' presence) ..................................................................... 35 ngre 2.2. Banding pattern for ederase (EST) isoenzymes visualized in a 7.5 % polyacrylamide native gel. Protein extracts (30 mg/Iane) from one, two, and three—week-Old Zea mays L. roots. Plants that received Glomus intraradices and/or an application of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and '+" presence) .................................................................................................. 37 Figure 2.3. Total peroxidase (POX) activity in corn roots followed by VAM fungal inoculation and fcrrnononetin application. The 100% POX activity for week 1, 2, and 3 ranges between 0.19 tO 0.25 0D,, s" mg" protein. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-' indicates absence and "+' presence). Each point is the mean Of five replicates. Vertical lines correspond to the standard error ....................................................... 40 Figure 3.1. Root colonization from three week-Old mycorrhizal (Glomus intraradices) Zea mays roots grown under 5 different soil P levels (P-1= 8; P2 = 24; P-3 = 41; P-4 = 74 and P-5 = 110 mg" Of soil) in the presence or absence Of exogenously applied formononetin. Plants that received the application Of fcrrnononetin are indicated as +FOR. Each point is the mean Of five replicates. Vertical lines correspond tO the standard error. +VAM-FOR (y=43.9-0.16x; r=‘0.83); +VAM+FOR (y=50.6-0.20x; r5088 .................................................................................................................. 62 Figure 3.2. Total acid phosphatase activity (ACP) from three week-Old mycorrhizal and non. mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8; P-2 = 24; P-3 = 41; P-4 = 74 and P-5 = 110 ug.g“ Of soil) in the presence or absence Of exogenously applied fcrrnononetin. The total ACP activity is expressed as pg Pi released ' min" mg" Of protein. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and '-I-' presence). Each point is the mean Of five replicates. Vertical lines correspond to the standard error ....................................................... 63 Figure 3.3. Total alkaline phosphatase activity (ALP) from three week-Old mycorrhizal and non- mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8; P-2 = 24; P-3 = 41; P4 = 74 and P-S = 110 mg" Of soil) in the presence or absence Of exogenously applied fomiononetin. The total ALP activity is expressed as pg Pi released min“ mg" Of protein. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and '+" presence). Each point is the mean of five replicates. Vertical lines correspond tO the standard error ....................................................... 65 Figure 3.4. Banding pattern for acid phosphatase (ACP) isoenzymes visualized in a 7.5 % polyacrylamide native gel. Protein extracts (30 mg/lane) from three-week-Old mycorrhizal and non-mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1 = 8; P-2 = 24; P-3 = 41; P-4 = 74 and P-5 =110 ug.g" Of soil) in the presence or absence Of exogenously applied fcrrnononetin. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-' indicates absence and '+' presence) ............................ 66 Figure 3.5. Banding pattern for NAD-malate dehydrogenase (MDH) Isoenzymes visualized in a 7.5 % polyacrylamide native gel. Protein extracts (30 mgllane) from three-week—old mycorrhizal and non-mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8; P2 = 24; P-3 = 41; P-4 = 74 and P-5 = 110 pg.g" Of soil) in the presence or absence Of exogenously applied fcrrnononetin. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-' indicates absence and '+' presence) ............................ 69 Figure 3.6. Banding pattern for esterase (EST) isoenzymes visualized in a 7.5 % polyacrylamide Figure 3. 7. native gel. Protein extracts (30 mg/lane) from three-week-Old mycorrhizal and non- mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8; P-2 = 24; P-3 = 41; P4 = 74 and P-5 = 110 pgg" Of soil) in the presence or absence Of exogenously applied fcrrnononetin. Plants that received Glomus intraradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and ‘+" presence) ..................................................................... 71 Total peroxidase (POX) activity from three week-Old mycorrhizal and non- mycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8; P2 = 24; PS = 41; P-4 = 74 and P-5 = 110 ug.g" Of soil) in the presence or absence Of exogenously applied fcrrnononetin. The total POX activity is expressed as OD“ 5" mg" protein. Plants that received Glomus intmradices and/or an application Of fcrrnononetin are indicated as VAM and FOR, respectively ('-' indicates absence and '+' presence). Each point is the mean Of five replicates. Vertical lines correspond to the standard error ............................................................................ 73 xi Figure 3.8. Figure 4.1. Figure 4.2. Figure 4.3. Figure 4.4. Figure 4.5. Figure 4.6. Banding pattern for peroxidase (POX) isoenzymes visualized in a 7.5 % polyacrylamide native gel. Protein extracts (30 mgllane) from three-week-old mycorrhizal and nonomycorrhizal Zea mays roots grown under 5 different soil P levels (P-1= 8: P-2 = 24; P-3 = 41; P4 = 74 and P-5 = 110 ug.g" Of soil) in the presence or absence Of exogenously applied fcrrnononetin. Plants that received Glomus intraradices and/or an application Of fonnononetin are indicated as VAM and FOR, respectively ('-' indicates absence and '+' presence) ............................ 74 StnIctures Of p-coumaric acid (3-(4-hydroxyphenyl)-2-propenic acid), p- hydroxybenzoic acid (4-hydroxy) and quercetin (3, 3', 4', 5, 7 - pentahydroxyflavone) ............................................................................................ 98 Growth and root colonization Of inoculated (Glomus intraradices) Clover plants as affected by different phenolic compounds applied once, at planting, at 0.25 and 1.0 mM solution concentrations (experiment A). Data represent mean Of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05) .................................................................. 102 Growth and root colonization Of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied 3 times, 7 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment 8). Data represent mean Of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05) ...................................................... 105 Growth and root colonization Of inoculated (Glomus intraradices) Clover plants as affected by different phenolic compounds applied 5 times, 4 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment C). Data represent mean Of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05) ...................................................... 108 Growth and root colonization Of inoculated (Glomus intraradices) sorghum plants as affected by different phenolic compounds applied 3 times, 7 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment D). Data represent mean Of 12 plants with standard errors. Means followed by the same letter within each graph are not significame different by LSD(p<0.05). ..................................................... 110 Growth and root colonization Of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied 5 times, 4 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment E). Data represent mean Of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05) ...................................................... 113 xii CHAPTER 1 INTRODUCTION Mycorrhizae Mycorrhizae are normally symbiotic, nonpathogenic associations between plant roots and certain SOiI fungi (Stahl, 1990). There are five broad groupings Of mycorrhizas: the vesicular-arbuscular mycorrhizas, the ectomycorrhizas, the ericaceous mycorrhizas, the ectendomycorrhizas and the orchidaceous mycorrhizas (Lewis, 1975). Each has distinct features Of morphology, anatomy, taxonomy Of compatible host plant and fungal taxonomy (Lewis, 1975). Vesicular-arbuscular mycorrhizae is the predominant type Of mycorrhizae. These fungi can colonize a vast taxonomic range Of plants in most ecosystems and is also the predominant type Of mycorrhizae formed with agricultural crops, tropical rainforest species, and nonforest ecosystems (Harley and Smith, 1983; Safir, 1987). Thus, the plants that do not form mycorrhizal symbioses are considered an exception in nature (Gerdemann, 1968; Newman and Reddel, 1987). The microsymbionts that form vesicular-arbuscular mycorrhizae belong to the taxonomic order Glomales and the sub-orders Glomineae and Gigasporineae in the Zygomycetes (Morton and Benny, 1990). There are six major genera Of the aproximately 150 species: Glomus, Gigaspcra, Acaulcspora, 2 Sclerocystis, Scutellispora, and Entrophospora belonging to the families Glomaceae, Acaulosporaceae, and Gigasporaceae (Morton and Benny, 1990). Germination process Most soils contain vesicular-arbuscular mycorrhizal (VAM) fungi. The spores Of the VAM fungi (clamydospcres) germinate and the hyphae elongate in the soil. In the presence Of a plant root, the infective hyphae originate from germinating spores. These hyphae attach to the root surface and form appressoria through which rOOt penetration takes place. However, if there are no plant roots in the vicinity hyphal growth ceases and the dormant state resumes, (Gianinazzi, 1991). Although VAM fungal spores possess the metabolic machinery and genetic information for germination and for initiation Of hyphal growth (Siqueira et al., 1985), continuous independent growth and sporulation, can only occur in the presence Of plant roots. Nevertheless, even without physical contact with plant roots, hyphal growth is stimulated (Becard and Piche, 1989a). Hyphal growth, branching, differentiation, and host penetration are affected by plant signals. Recently, studies have indicated that root exudates contain factors that enable the fungus tO use its spore reserves and subsequently trigger arbuscule formation (Becard and Piche, 1989a). Besides rOOt exudates, suspension- cultured cells and cell products (Carr et al.,1985; Paula and Siqueira, 1990) and volatiles, such as CO,, apparently are involved in signalling processes. Furthermore, the possible signalling between roots and hyphae is thought to 3 occur only within a few millimeters distance between the hyphal tip and the root (Gianinazzi-Pearson et al., 1990), since the direction Of hyphal growth occurs randomly in the soil. Spores Of most species of VAM fungi can readily germinate in vitro, but subsequent hyphal development is limited (Becard and Piche, 1989a). Hyphal growth can continue over a short time interval and then ceases before there is total depletion Of spore reserves. Becard and Piche (1989a) proposed two mechanisms that involve the role Of the root in stimulating fungal growth: 1) hyphal growth from a germinating spore initially depends on nutritional reserves in the spore, and this growth is Slow and limited, and roots can stimulate hyphal growth, either through direct activation or by removal Of inhibitors. This phase of growth is still dependent on and is limited by spore reserves; and, 2) the developmnent Of arbuscules enables the fungus tO utilize the rOOt as a carbon (C) source. This phase is exclusively root dependent. Thus, the mechanisms responsible for the VAM Obligative biotrophy may involve a combination Of nutritional, physical and genetic factors, yet to be elucidated (Siqueira, 1987). Establishment of mycorrhizae Germinating hyphae proliferate close to the roots, and immediately after contact with their hosts, VAM fungi form more-or-less well-defined appressoria (Gianinazzi, 1991), which indicates that some kind Of recognition occurs at this early stage Of VAM formation. Fungal hyphae penetrate from the appressorium into the outer layer Of root tissues, both inter- and intracellularly. (Bonfante- 4 FasolO, 1984). The fungus subsequently penetrates into deeper layers Of the root cortex, where both intercellular and intracellular hyphae branch to form vesicles, the storage sites for fungal lipids (Cooper, 1984). Intracellular hyphae also differentiate into ephemeral, haustoria-like structures called arbuscules, with high surface-tO-volume ratios. Arbuscules are a major site Of nutrient exchange between the macro- and microsymbiont (Bonfante-Fasolo, 1988). Increased H’- ATPase activity in arbuscular membranes, as well as in pen-arbuscular membranes, indicate high transport activity (Gianinazzi-Pearson, 1991). Arbuscule development is also accompained by dramatic increases in host plasmalemma formation, resulting in a perisymbiotic membrane that surrounds all branches Of the developing arbuscule (Alexander et al., 1988). Infection progresses along growing roots in a dynamic fashion (Jacquelinet-Jeanmougin et al., 1988). VAM hyphal growth stops in cortical tissues; hyphae never penetrate meristems or the central cylinders Of rOOts (Alexander et al., 1988; Jacquelinet-Jeanmougin er al., 1988). In reality, each arbuscule formed by a branch Of the fungus growing into parenchymal cells represents a terminal structure Of fungal development (Alexander et al., 1988). Arbuscules die after a few days of endocellular life (4 to 10 days) and are degraded by the plant. At this time the host cells begin tO resemble normal parenchymatous cortical cells (Jacquelinet-Jeanmougin et al., 1988). A permanent biotrophic phase between the two symbionts is maintained only because arbuscule death does not affect the development Of residual mycelium, which continues to grow and form arbuscules in other parenchymal cells. Plant reactions to VAM fungi Fungal development within the host is likely modulated by host hydrolytic enzymes (Gianinazzi, 1991) although VAM fungi could also trigger several host- defense responses. Chitinase and peroxidase enzymes are generally considered as defense enzymes in plant-pathogen interactions (Van Loon, 1986). Peroxidases likely make the host-cell wall more resistant to pathogen penetration, while Chitinase degrades chitin, a component Of most fungal cell walls (Boiler at al., 1983). In mycorrhizal leek, peroxidase and Chitinase activitities increased above levels in non-inoculated roots during the first few days Of root colonization and then decreased below those Of non-mycorrhizal roots at later stages Of the symbiosis (Spanu and Bonfante-Fasolo, 1988; Spanu et al., 1989). In these studies, both peroxidase and Chitinase activities peaked during mycorrhizal penetration and the early stages Of root colonization. Using histochemical techniques, this enzyme activity has been localized in the epidermal and hypodermal cells Of mycorrhizal leek and onion roots and in the middle Iamella around growing intercellular hyphae in the root cortex, but it is not present in cells containning intracellular arbuscular hyphae (Gianinazzi, 1990). In addtion, it has been shown that phytoalexins accumulate tO a minor extent in soybean mycorrhizal roots (Morandi et al., 1984; Wyss et al., 1991). Therefore, VAM fungi elicit a weak non-Specific plant defense reaction, possibly facilitating the establishment Of VAM symbiosis. Plant growth responses to VAM symbiosis VAM fungi contribute tO plant growth by enhancing nutrient uptake and translocation, while the host provides carbon (C) compounds necessary for fungal growth (Jakobsen and Rosendahl, 1990). In general, mycorrhizal plants have higher rates Of growth than non-mycorrhizal plants, when growing in soils low in available phosphorus (P). Often, effects on plant growth are not apparent for several weeks after VAM germination. Factors that contribute tO this delay include a lag phase before the onset Of rapid fungal colonization Of the roots and competition for photosynthate between the fungi and plant hosts (Smith and Gianinazzi-Pearson, 1988). However, it may also be true that certain species Of VAM fungi may occasionally reduce relative plant growth (Johnson et al., 1992), particularly when high fertilization rates are used (Hepper, 1983). Although the assimilation Of a number of mineral nutrients is enhanced by VAM fungal colonization, P plays a prominent role. Once an endomycorrhizal symbiosis is established, the major fungal contribution is nutrient uptake and translocation. P uptake increases in mycorrhizae due tO the efficiency with which the soil profile is exploited. Extraradical hyphae can extend significantly beyond root and root hair depletion zone (Hayman, 1983). In addition, VAM fungi is capable Of entering soil microaggregates, which roots are not able to penetrate (Pacovsky, 1989). For nonmobile nutrients such as P and Zn, rOOt growth, root hair development, and the initial ion concentration in the soil solution determine the rate Of uptake (Clarkson, 1985). Mycorrhizal modification Of the root’s nutrient-uptake properties depends upon (a) development Of extramatrical 7 hyphae in soil, (b) hyphal absorption Of phosphate, (C) translocation Of P through hyphae over considerable distances, and (d) transfer Of P from the fungus to the root cells (Rhodes and Gerdeman, 1975). VAM fungi are able to absorb phosphorus at lower solution concentrations than the non-VAM roots (Hayman and Mosse, 1972). Recently (Jayachandran et al, 1992), demonstrated that VAM fungi have the ability to solubilize organic forms Of phosphate, which are not accessible to non-VAM roots. The initial SOil P availability is also very important in determining the outcome Of the symbiosis. Generally, under high soil available P, plants will have the ability tO reduce mycorrhizal infection (Hepper, 1983). Various hypothesis have been suggested: membrane permeability (Ratnayake et al., 1978), quantity Of root exudates ( Graham et al., 1981) or quality Of rOOt exudates (Elias and Safir, 1987). Under low phosphorus nutrition, root cell membranes have higher permeability thereby allowing increased loss Of root metabolites, which is beneficial tO colonization by VAM fungi. Improved phosphorus nutrition Of the plant by VAM subsequently leads tO reduced membrane permeability and a reduction in root metabolites. In this condition, mycorrhizal root colonization can become deleterious to plant growth, by requiring host carbohydrates, without a concomitant enhancement in plant mineral nutrition, especially P. In addition tO increased nutrition, mycorrhizal plants also can have increased resistance to root pathogens, including nematodes (Rosendahl and Rosendahl, 1990), increased tolerance tO drought (Nelsen and Safir, 1982) and salt stress (Rosendahl and Rosendahl, 1991), and heavy metal toxicity (Read, 1986). Applications of isozymes In VAM research Recently, isozyme analysis Of VA mycorrhizae or fungi has been used in taxonomy, identification and quantification Of rOOt infections, and in ecological and biochemical studies. The taxonomy Of VAM fungi is a difficult task due our inability tO grow these fungi in chemically defined media in the absence Of living roots. At the generic level, taxonomy Of VAM fungi is based primarily on the color, form, size, and type Of hyphal attachment (Trappe and Schenck, 1982). At the species level, the main diagnostic characteristics used are the structure Of the spore cell wall, its thickness and ornamentation (T rappe and Schenck, 1982). The use Of murographs have also been suggested for identification and classification Of VAM fungi (Walker, 1983), whereas the visual characteristics Of resting spores has been considered less reliable and is only performed reliably by specialized taxonomists. Furthermore, VAM fungi Show considerable intraspecific variation, an indication Of a high degree Of physiological adaptation Of the fungi tO a broad range Of environmental conditions (Rosendahl and Sen, 1992). Isozyme analysis has been one method used in taxonomic studies Of several controversial groups Of VAM fungi, particularly Glomus spp. (Sen and Hepper, 1986; Hepper et al., 1986; Hepper et al., 1988a; Hepper et al., 1988b). Sen and Hepper (1986) characterized several Glomus species by selective enzyme staining following electrophoretic separation on polyacrylamide gels Of fungal proteins extracted from resting spores. Although this method has shown to be sensitive to the differences at the species level and was reproducible between assays, Morton (1988) pointed out the importance Of combining morphological and chemotaxonomic criteria when identifying VAM fungi. Several methods for quantifying VAM colonization have been described. The colonization by VAM fungi can be assessed microscopically after clearing the roots with potassium hydroxide and staining (Phillips and Hayman, 1970) with either acid fuchsin or trypan blue. Fungal structures can thus be easily identified under a microscope or a dissecting scope, using the line intersect method (Kormanik and Mcgraw, 1982), in which the number Of root pieces bearing fungal structures colonization is measured as a percent Of root length colonized. Also, colonization can be assessed biochemically, in which glucosamine content Of the roots is measured colorimetrically (Ride and Drysdaly, 1972). These methods are commonly used, but do not distinguish living from dead fungus. Rosendahl et al. (1989) quantified VAM fungi colonizing cucumber roots using diagnostic enzymes. They found a positive correlation between selected fungal enzymes and glucosamine content, and suggested that fungal biomass can be used tO estimate the colonization within the roots. Histochemical analysis Of succinate dehydrogenase has been used to measure the metabolic activity Of VAM hyphae within the roots. This method detects the proportion Of infected roots in which the fungus is active (“alive”) (Ocampo and Barea, 1985; Smith and Gianinazzi-Pearson, 1990). However, this method does not appear tO be indicative Of the efficiency Of VAM symbiosis, in terms Of nutrient uptake or plant growth stimulation. Thus, another 10 histochemical method involving the enzyme alkaline phosphatase was investigated tO whether this enzyme could provide a useful physiological marker for the efficiency Of the fungus in terms Of plant growth improvement (T isserant et al., 1993). At early stages Of infection, only a small proportion Of living intraradical mycelium had alkaline phosphatase activity, but this activity increased greatly, immediately before the mycorrhizal growth response Of the host plant. They suggested that alkaline phosphatase could provide a useful marker for analysing the symbiotic efficiency Of VAM infections. Identification Of individual and combined infections in roots Of leek and maize (Hepper et al., 1986) has now been made possible through isozyme analysis. This technique can also be used to determine the contribution Of a Single species in a mixed population. Enzymes involved in the primary metabolic processes such as glucose-6-phosphate dehydrogenase, glutamate oxaloacetate transaminase, glutamate dehydrogenase, esterase, peptidase, and malate dehydrogenase, have also been used for diagnostic purposes. In addition, isozyme patterns can provide a valuable technique for ecological studies Of mycorrhizal fungi. For example, diagnostic fungal isozyme analysis has been used for monitoring the outcome of an inoculation programme by dectecting the presence Of the introduced species in the host roots (Hepper et al., 1988). lntemal hyphae have been removed from colonized roots and analyzed. However, enzymic digestion of the plant roots is necessary. The enzymes pectinase and cellulase are used tO hydrolyse the pectic layer between cells and then remove cellulosic cell walls. Theoretically, the fungal structures should 11 remain intact since fungal walls are composed Of chitin. However, McGee and Smith (1990) indicated that there is damage to the fungal structures and effects on enzyme activity during digestion. Therefore, they emphasized that isozyme patterns Obtained from internal hyphae, Should not be regarded as highly reliable. The establishment Of a mycorrhizae leads tO a complex sequence Of events between the fungus and the host plant, before a functional symbiosis takes place ( Bonfante-Fasclo and GripiollO, 1982; Bonfante-FaSOIO et al., 1986). Little is known about the physiology Of the establishment Of mycorrhiza at early stages, compared tO mature endomycorrhizal symbioses (Wyss, 1991). The process Of infection requires several steps and necessitates the penetration Of hyphae along and through cell walls (Gianinazzi-Pearson, 1991). The use Of selected isozymes could thus provide additional biochemical inforrnaticn concerning the host-fungus relationship in the early establishment Of the symbiosis. Role of phenolics in VAM symbioses Phenolic compounds, the most important class Of plant secondary metabolites, are released in the soil as leachates, exudates or by decaying residues (Siqueira et al., 1991a). Ferulic, p-coumaric, and p-hydroxybenzoic acids are the predominant phenolic acids in the root Of most species. Other phenolics, such as flavonoids, are also present, but usually in low concentartions (Putman, 1988). These phenolics have been isolated from a variety Of soils and their concentrations depend on the extraction procedure (Dalton et al., 1987; LOdhi, 12 1975), soil type (Whitehead, 1964; Wang et al., 1967; Dalton, 1989), sampling time (Lodhi, 1975), vegetation type (Guenzi and McCalIa, 1966; Wang et al., 1967), and many other factors. Guenzi and MCCaIIa (1966) estimated that the residue from a single crop Of sorghum could return about 100 Kg'ha"'year" Of p- coumaric acid tO the soil. In sugarcane field soil, 30.3, 6.9, and 6.5 moles 100 g" of soil for p-C0umaric, vanillic and ferulic acids, respectively, were found (Whitehead, 1964). Also, the amounts Of p-coumaric, ferulic and caffeic acids in January soil samples were higher in the 0-15 cm level than in the 15-30 cm level; however, the amounts in the 0-15 cm layer consistently decreased from January to September, while in the 15-30 cm layer, the amount Of these phenolics increased (Lodhi, 1975). These Changes Show that phenolics in soil-plant systems probably undergo continuous cycles Of deposition, decomposition, plant uptake, leaching, and chemical immobilization (Siqueira et al., 1991a). Thus, at certain times phenolics may function as allelopathic agents, particularly when environmental factors favor their accumulation, or release, in the soil. It also may be possible that low levels Of these compounds, through continuous production and decomposition, are effective as allelopathic agents without ever building up tO high concentrations in the soil (Dalton, 1989). These molecules which are free in the soil solution are in intimate contact with living soil organisms and plant roots and therefore influence their growth and activity. In fact the continuous input Of these compounds may over time change the microbial population Of the soil (Blum and Shafer, 1988) and indirectly affect plant growth. 13 Several studies have suggested the involvement Of autotoxic asparagus Chemicals (Hartung, 1987; Young, 1984). Asparagus has also been shown to respond favorably to VAM inoculation (Powell and Bagyaraj, 1983). Ferulic acid, one allelochemical isolated from asparagus tissue (Hartung, 1987), has been Observed to reduce asparagus VAM colonization and plant growth with increasing concentrations (Wacker et al., 1990b). Also, there was a Shift in the VAM species composition, which was correlated with field age (Wacker et al.,1990a). In addition, monocropping system Of corn (Vivekanandan and Fixen, 1991) and soybean (Johnson et al., 1992) Significantly decreased plant growth, tissue P concentrations, and yield compared tO those grown in rotation. They suggested that shift in VAM populations occurs under monocropping, and the most rapidly growing and sporulating fungal species (inferior mutualists or even parasitics) prevail. It is now known that the perennial and monocropping systems allow the build up Of phenolic compounds in the soil (Siqueira et al., 1991a). Thus, phenolic compounds may play an important role in the establishment Of mycorrhizal associations in the field. Although it is generally accepted that VAM fungi are non-specific in their selection Of hosts (Harley, 1985), different degrees Of mycorrhizal dependency Of the host plant have been found, which depend on the plant’s nutrient requirement, root distribution and the prevailing SOil fertility (Plenchette et al., 1983). Differences in colonization potential Of various cultivars Of wheat (Azcon and Ocampo, 1981), pearl millet (Krishna et al., 1982), and soybean (Heckman and Angle, 1987) have been Observed. In addition, different Glomus species 14 infected similar root lengths but the degrees Of stimulated root growth and interaction with phosphate uptake were different (Estaun et al., 1987). It has also been suggested that particular host-fungus combinations may be more symbiotically effective, if the physiology and growth rates Of the symbionts are better matched (Smith and Gianinazzi-Pearson, 1988). This may involve a recognition process between the fungus and host through specific chemical signals, probably Of phenolic nature (Siqueira et al., 1991 a). In spite Of intensive basic and applied research, the growth Of VAM fungi in pure culture is still a Challenge for the future. Hyphal growth is stimulated by the presence Of roots (Becard and Piche, 1989b) or root exudates from plants (Elias and Safir, 1987). As in another symbiotic systems (Lynn and Chang, 1990), phenolics may also play a role as signal molecules in VAM establishment. The flavonones, hesperetin and naringerin, and the flavone, apigenin, stimulated hyphal growth in vitrO (Gianinazzi-Pearson et al., 1989). The flavonoid, quercetin, stimulated spore germination, hyphal elongation and branching (Tsai et al., 1991) Of Glomus etunicatum (Tsai et al., 1991), Gigaspora margarita (Becard et al., 1992), and recently, Gigaspora gigantea (Baptiste and Siqueira, 1994). In addition, the isoflavone formononetin, isolated and identified from Clover roots (Nair et al., 1991) promoted the hyphal growth in an in vitrO assay. Furthermore, the stimulatory effect of formononetin on white clover VAM formation and growth was confirmed (Siqueira et al., 1991). The application Of formononetin also reduced herbicide injury (Siqueira et al., 1991c), and 15 increased plant growth and yield Of com and soybean in the field (Siqueira et al., 1992). Although the stimulatory effects Of fcrrnononetin on mycorrhizal symbiosis have been Observed, the physiological reasons for this stimulation aspects involving VAM fungi and plant roots have not been investigated. In Chapter 2, the effect Of exogenously applied fcrrnononetin on the alteration Of some isozyme activities (malate dehydrogenase, esterase and peroxidase) were monitored during the early stages Of a Zea-Glomus symbiosis. In Chapter 3, plants were grown under different levels Of soil avaible P, and the isozyme (acid phosphatase, alkaline phosphatase, malate dehydrogenase, esterase and peroxidase) activities Of a Zea-Glomus symbiosis were studied at 3 weeks Of growth. In Chapter 4, the possible effects Of exogenously applied phenolic compounds (p-coumaric acid, p-hydroxybenzoic acid, and quercetin) on plant growth and root colonization of clover and sorghum plants were investigated, using different concentrations and soil applications. LIST OF REFERENCES Alexander, T., Meier, R., Toth, R. and Weber, H. G. (1988) Dynamics Of arbuscule development and degeneration in mycorrhizas Of Triticum aesticum L. and Avena sativa L with reference to Zea mays L New Phytologist 110, 363-370. 16 Azcon, R. and Ocampo, J. A (1981) Factors affecting the vesicular-arbuscular infection and mycorrhizal dependency Of thirteen wheat cultivars. New Phytologist 87, 677-685. Baptiste, M. J. and Siqueira, J. O. 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L (1990) Revised Classification Of arbuscular mycorrhizal fungi (Zygomycetes): a new order, Glomales two new suborders, 21 Glomineae and Gigasporineae, and two new families, Acaulosporaceae and Gigasporaceae, with en emendation Of Glomaceae, Mycotaxon 37, 471. Nair, M. G., Safir, G. R. and Siqueira, J. O. (1991) Isolation and identification Of vesicular-arbuscular mycorrhiza stimulatory compounds from Clover (Trifolium repens) root. Applied and En vironmental Microbiology 57 , 434-439. Nelsen C. E. and Safir G. R. (1982) Increased drought tolerance Of mycorrhizal onion plants caused by improved phosphorus nutrition. Planta 154, 407-413. Newman, E. l. and Reddel, P. (1987) The distribution Of mycorrhizas among families Of vascular plants. New Phytologist 106, 745-751. OcampO J. A and Barea J. M. (1985) Effect Of carbonate herbicides on VA mycorrhizal infection and plant growth. Plant and Soil 85, 135-141. Pacovsky R. S. (1989) Carbohydrate, protein and amino acid status Of Glycine- Glomus- Bradyrhizobium symbiosis. Physiologia Plantarum 75, 346-354. Pacovsky R.S. (1989b) Metabolic differences in Zea-GIomus-Azospirillum symbioses. Soil Biology and Biochemistry 21, 953-960. Paula, M. A and Siqueira, J. O. (1990) Stimulation Of hyphal growth Of the VA mycorrhizal fungus Gigaspora margarita by suspension-cultured Pueraria phaseoloides cells and cells products. New Physiologist 115, 69-75. Phillips, J. M. and Hayrnen D. S. (1970) Improved procedure for clearing roots, and staining parasitic end vesicular- arbuscular mycorrhizal fungi for rapid assessment Of infection. Transactions of the British Mycological Society 55, 158- 161. Plenchette, C., Furlon, V. and Fortin, J.A (1982) Effects Of different endomycorrhizal fungi on five host plants grown on calcined montrnorillcnite clay. Joumal Arnen'can Society of Horticultural Science 107: 535-538. Reed, D. J. (1986) Non-nutritional effects Of mycorrhizal infection. In V. Gianinazzi (eds) Physiological and Geneticel Aspects Of Mycorrhizae. INRA Press, Paris. Ride, l. and Drysdele, R. B. (1972) A rapid method for the estimation Of filamentous fungi. Physiological Plant Pathology 2, 7-15. Rosendahl, ON. and Rosendahl, S. (1990) The role Of vesicular-arbuscular mycorrhiza in controlling damping-Off and growth reduction in cucumber caused by Phyfltium ultimum. Symbiosis 9, 363-366. Rosendahl, ON. and Rosendahl, S. (1991) Influence Of veisculer-arbuscular mycorrhizal fungi (Glomus spp.) on the response Of cucumber (Cucumis sativus L) to salt stress. Enviromental and Experimental Botany 31, 313-318. Rosendahl, S. Sen, R., Hepper, CM. and Azcon-Aguilar, C. (1989) Quantification of three vesicular-arbuscular mycorrhizal fungi (Glomus spp) in roots Of leek (Allium porrum) on the basis of activity Of diagnostic enzymes after polyacrylamide gel electrophoresis. 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CHAPTER 2 EXPRESSION OF ISOENZYMES ALTERED BY BOTH Glomus intraradices COLONIZATION AND FORMONONETIN APPLICATION IN CORN (Zea mays L.) ROOTS INTRODUCTION Although there are many studies on mature endomycorrhizal symbioses. comparatively little is known about the physiology Of the interaction between host and fungus at the earliest stages Of colonization (Wyss et al., 1991). A complex sequence Of events occurs between fungal hyphae and host cells before a functional symbiosis takes place (Bonfante-Fasolo and Grippiolo, 1982; Bonfante-Fesolo and Gianinazzi-Pearson, 1986). The discovery and Characterization Of specific physiological alterations could be used to monitor and assess the early interplay between host and endophyte, and this would allow a greater insight into the recognition process that occurs with both organisms. In a functioning mycorrhiza, there are changes at the organismal, tissue, cellular, and molecular levels (Bonfante-Fasolo, 1987). In maize, the presence Of endomycorrhizal fungi also shifts the metabolism and biochemical partitioning Of assimilates in a manner that influences the physiology Of the entire plant (Pacovsky, 1989b). It has been Observed that glycolytic enzymes and various dehydrogenases 26 27 have been stimulated in arbuscular mycorrhizal (AM) plants (Dehne, 1986), and the distrbution Of carbohydrates, amino acids and lipids in Glycine-Glomus symbioses changed following fungal colonization (Pacovsky, 1989a). Also, the discovery Of a new Class Of soluble proteins inside AM roots, the endomycorrhizflns (Dumas et al., 1989), suggested that there is a marked alteration Of gene expression as a result Of fungal colonization (Pacovsky, 1989a). Specific isoflavonoids may act as early plant Signals that influence the activity Of mycorrhizal fungi (Siqueira at al., 1991). Plant phenolic compounds are important signal molecules in the Rhizobium-legume symbiosis, in Agrobacterium-plant infections and in growth Of certain parasitic weeds (Lynn and Chang, 1990). The isoflavone formononetin, a secondary plant metabolite produced by stressed Clover plants (Nair et al., 1991), has been shown tO increase the rate Of vesicular- erbuscular mycorrhizal (VAM) colonization on inoculated clover roots at early stages Of the symbiosis (Siqueira at al., 1991). This increase in the rate Of colonization undoubtely has an impact on the physiology Of developing mycorrhizal associations. Forrnononetin may be acting systemically in the plant (VOIpin et al., 1994), and if SO, it may affect rapidly growing roots lower in the soil making these roots more suitable for fungal colonization. Forrnononetin concentration consistently increased in alfalfa roots when VAM fungal spores were present in the rhizosphere or when mycorrhizal hyphae had colonized the root cortex. However, fcrrnononetin was not acting as a signal molecule since it increased even in P-Sufficient plants that would not support high levels Of infection by the endophyte (VOlpin et al., 1994). Endomycorrhizal fungi rely on carbon from the plant for development and growth. Jakobsen and Rosendahl (1990) demonstrated that 43% of photoassimilated “C was allocated below-ground and 27% was utilized for respiration in cucumber mycorrhizae. These values for C utilization were much higher than in non-VAM plants. Similarly, additional C export tO the root and higher rates Of root respiration has confirmed the greater C demand Of VAM compared to a non-VAM root (Snellgrove et al., 1982). Since enzymes are the regulatory molecules in biochemical pathways, including carbon and cellular metabolism, we studied a number Of enzymes with well- characterized physiological roles. Enzymes involved in carbon turnover (malate dehydrogenase, MDH; esterase, EST), and in plant structure and defense (peroxidase, POX) were screened for possible physiological alterations following VAM colonization. We examined the variation in certain isozyme activities during the earliest stages Of colonization (first three weeks Of growth) in VAM or non-VAM corn roots, treated with fcrrnononetin or not and we present evidence that some isozymes can be used to monitor and assess the early host-endophyte interactions in the Zea-Glomus symbiosis. MATERIALS AND METHODS Experimental design Corn plants (Zea mays L cv. Great Lakes-hybrid 582) were grown in soiVsand mixture for 1-, 2-, and 3-weeks post-emergence. There were four treatments, derived from a 2 X 2 factorial where mycorrhizal (VAM) status or fcrrnononetin (FOR) presence were the main effects. Treatments consisted of: control (-VAM-FOR), either fcrrnononetin (-VAM+FOR) alone or Glomus intraradices Schenck & Smith (+VAM-FOR) alone, or both formononetin and G. intraradices (+VAM+FOR). There were five replications per treatment for a total of 60 plants. Growth conditions Three corn seeds were sown in 1.6 kilograms Of a mixture Of steam-sterilized greenhouse top soil and silica sand (1:1, V:V), and plants were thinned to one seedling per pot post-germination to achieve a uniform stand. Soil analysis indicated a pH Of 7.8, and 117 mg N03, 0.2 mg P, 20 mg K, 271 mg Ca, 71 mg Mg, 20 mg Mn, 1.3 mg Zn, and 5.0 mg Cu per kg soil. Half Of the pots containing this substrate were inoculated with spores Of Glomus intraradices isolated from a commercial clay matrix (Nutralink, Native Plants lnc., Salt Lake City, UT) by wet sieving through 420 mm and 38 mm mesh sieves (approximately 6400 spores used per pot). The other pots were left non-inoculated. Soil inoculated with G. intraradices was throughly and uniformly mixed to give a final concentration of approximately 4 spores 9“. Then to half Of the pots, 200 ml Of a 4 (1ng solution of fcrrnononetin (Rhizotech lnc., Hopewell, NJ) was added to the soil at onset Of the experiment to complete the 2 X 2 factorial design. Plants were grown in a greenhouse, at Michigan State University, East Lansing, MI, where the plants received, in addition to sunlight, supplemental light from sodium-vapor lamps (illumination approximately 650 mmol In”2 s") for 16 h per day. Plants received 100 ml of nutrient solution every other day, beginning 2 days after seedling emergence. The nutrient solution contained 0.25 mM CeCL, 0.5 mM K80" 0.5 mM K,HPO,, 2.5 mM NH,NO., 1.0 mM (NH),SO,, 25 mM H,BO,, 20 mM FeNaEDTA, 2.0 mM ZnSO,, 0.5 m_M_ CuSO, and 0.4 mM Na,MOO, (modified from Pacovsky and Fuller, 1988). Nutrient solution and distilled water were applied to the bottom Of each pot where there was a catchment dish. Evduatlons and assays At each harvest, corn roots were carefully separated from the soil and washed with distilled water. Fresh weights were measured on both shoots and roots. Shoots were oven dried at 65'C until a constant weight was obtained (3 days). Roots were wrapped in aluminium foil, immediately frozen in liquid nitrogen, and stored at -20°C until use. A portion Of the root sample was thawed, Cleared and stained with trypan blue (Phillips and Hayman, 1970), and percentage of root segments colonized by G. intraradices was estimated by the line-intersect method (Korrnanik and McGraw, 1982). The remainder of the frozen roots were then placed in a cooled mortar containing liquid N, and were ground with pestle in the presence of liquid N2 until finely powdered. Preparation of root extracts Pulverized root tissue was mixed with grinding buffer (0.75 ml 9" fresh root) containning 50 mM Tris-HCI pH 7.0, 3.0 mM ethylenediamine tetraecetic acid, 2.5 mM dithiolthreitol, 250 mM sucrose, 50 mM NaCl, 2mM phenylmethylsulfonyl fluoride, and 2 mM N-ethyl-maleimide (Pacovsky, 1989a). Cooled samples were 31 then homogenized with a tissue grinder (T ekmer, Cincinnati, OH), followed by centrifugation for 10 min at 12,000 x g at 4‘0 (Marathon 21K/BR, Fisher Scientific, Chicago, IL). The supemetants were collected and stored in microcentrifuge tubes at -20’C until use. Bradford's (1976) dye-binding assay was used to determine the protein concentration in each sample using bovine y-globulin as a standard (Biorad, Richmond, CA), and root protein content was calculated using root moisture content, fresh weight and extract volume for each sample. Polyacrylamide native gel electrophoresis Samples were prepared for discontinuous native polyacrylamide gel electrophoresis (PAGE) using a 4 % stacking gel and a 7.5 % separating gel according to Davis (1964). Thirty pg of total root protein was loaded per single well. Malate dehydrogenase (MDH), esterase (EST), and peroxidase (POX) isoenzymes were visualized using standard activity-staining procedures (Shaw and Prasad, 1 970). Total peroxidase activity measurements Measurements of total peroxidase activity were determined spectrophotometrically following a modified version Of Ridge and Osborne (1970). Ten pg Of protein from each sample was added to 4 ml of 11.36 mM H20. in 8 mM phosphate buffer (pH 7.0) and 1 ml Of 1.96 mM guaiacol. Optical density at 480 nm was recorded every 30 seconds for 10 minutes. Five replicates from each treatment at each harvest were assayed. Total peroxidase activity was expressed as OD“, 5" mg" protein. Percent activity was calculated based on the difference between the untreated control and each treatment mean. Statistical analysis The experiment (2 X 2 factorial with 5 replications per treatment) was repeated three times, and the results for plant growth, root colonization, peroxidase essay and isoenzyme patterns were essentially the same. Dry weight, root colonization, root protein content, and total peroxidase activity data, from one experiment, were subjected to a complete analysis Of variance. Treatment means were compared by least significant difference (LSD) test at P<0.05, while root colonization means were compared by Student’s t-test (P < 0.05). RESULTS Growth and colonization Shoot dry weights of +VAM-FOR and +VAM+FOR were greater than -VAM+FOR plants at week 1 (Table 2.1). At 2 weeks Of growth, only +VAM+FOR shoot dry weights were significantly (P<0.05) higher than controls (-VAM-FOR). The positive effect Of VAM colonization on shoot dry weight was observed at 3 weeks of growth. Root dry weights differed only at week 2 between NAM-FOR and +VAM+FOR roots (T able 2.1 ). Root colonization by G. intraradices increased in the presence of fcrrnononetin both at weeks 2 and 3 (Table 2.1). A dramatic increase in VAM colonization was Table 2.1. Plant growth and root colonization of corn plants at 1, 2 and 3 weeks of growth following Glomus intraradices (+VAM) inoculation or fcrrnononetin (+FOR) application. Plants were harvested at 1, 2, and 3 weeks of growth. Treatments Shoot dry Root dry Root' weight weight colonization (9) (9) (°/-) Week 1 - VAM - FOR 0.22 ab 0.16 a 0 -VAM+ FOR 0.18b 0.16a 0 +VAM - FOR 0.25 a 0.19 a 1 a +VAM+ FOR 0.25 a 0.19 a 2 a Week 2 - VAM - FOR 0.49 m 0.39 m 0 - VAM-I- FOR 0.52 lm 0.40 lm 0 +VAM - FOR 0.58 lm 0.47 Im 20 m +VAM+ FOR 0.62 l 0.48 I 36 l Week 3 - VAM - FOR 1.36 z 1.51 z 0 - VAM-I- FOR 1.29 z 1.45 z 0 +VAM - FOR 1.74 y 1.74 z 52 y +VAM+ FOR 1.74 y 1.85 z 60 z Means ( 5 replicates per treatment) followed by the same letter within a column are not significamly different by LSDm test. ‘ Means were compared using Student’s t-test at P < 0.05 34 observed at week 2 when % colonization in +VAM+FOR roots was nearly twice as high as +VAM-FOR roots. However, by the third week this difference was only 15 % greater for +VAM+FOR plants compared to +VAM-FOR plants. MAD-malate dehydrogenase isozymes Eleven isozymes bands were detected in the NAB-dependent malate dehydrogenase (MDH) activity stained gels (Figure 2.1) (seven major bends, MDH4 to 11, and three minor bands MDH1 to 3). Among those, two malate dehydrogenase isoenzymes (MDH3, MDH4) demonstrated higher activity in VAM- colonized corn roots at 3 weeks (Figure 2.1, lanes 7, 8). The second isozyme, MDH4 was expressed more strongly in mycorrhizae compared to MDH3. Mycorrhizal roots treated with fcrrnononetin showed slightly stronger intensities for MDH3 and MDH4 bands at 3 weeks compared to mycorrhizae alone (lanes 7, 8). MDH3 and MDH4 bands did not appear in one-week-Old roots where there was only trace of VAM colonization (lanes 3 and 4), while these two isoforms were only weakly expressed in VAM roots at 2 weeks (date not shown). Esterase Isozymes Eleven bands in the esterase (EST) activity-stained gels were Observed (Figure 2.2, seven major bands and four minor [EST 4, 5, 7, 8] bands). Mycorrhizal colonization caused an increase in the activity of four esterase isozymes (EST1, EST2, EST3 and EST5) at two weeks (Figure 2.2, lanes 7, 8). In the presence of fcrrnononetin, the expression of these isoenzymes was further enhanced (lane 8). Figure 2.1. Banding pattern for MAD-malate dehydrogenase (MDH) isoenzymes visualized it a 7.5 % polyacrylamide native gel. Protein extracts (30 ugllane) from one, two, and three-week-Old Zea mays roots. Plantsthat received Glomusintraradicesandloran application Offormononet'nare 'ncicetedes VAM and FOR, respectively ('-" Indicates absence and '+" presence). MALATE DEHYDROGENASE 1 WEEK 3 WEEKS 1 2 3 4 5 6 7 8 Rm Rm 016 ‘MDHI MDH2 0.22 026 ‘ . - MDH3 MDH4 031 0'34 I MDH5 MDH6 0'36 0'38 MDH7 MDH8 0.41 0.43 .MDHg - 0.49 MDH11 MDHIO 0.47 VAM ——++ - - +1“ VAM FOR-+-+ -+—+FOR 37 Flgure2.2.Bandhgpattanforesterase(EST)eoenzwnesvetaizedha7.5%pdyeayhmide native gel. Proteinextreds(30 pgllane)fromone, two,andthree-week-old Zea mays roots.Plants thatreceived Gbmushhamdbesandlmenapplicetbnoftonmtethemidcatedasvmw FOR, respectively ('-' indcates finance and '+' presence). 2 WEEKS 3 WEEKS Rm 9 10 11 12 0.16 024 ”O20 0.40 0.50 0.61 0'57 0.67 0.64 0.72 0'69 VAM_--+T; —-'++ --++VAM Tote At 3 weeks, when there was only 15 % difference in root colonization between +VAM-FOR and +VAM+FOR roots, all treatments showed similar levels of EST1, 2, 3, and 5 activities (lanes 9-12). Peroxidase Isozymes Twelve peroxidase (POX) isozymes were visualized in our native PAGE (not shown). In general, the highest activities for the various POX isozymes was seen at week 2. It did not appear as if a single isozyme was differentially altered by either VAM inoculation or fcrrnononetin application. As reported before (Bronner et al., 1991), some of the peroxidase isozymes assayed with guaiacol might not have been faithfully detected in our gels where o-dianisidin was used as substrate. Total peroxidase To quantify the influence of colonization or fcrrnononetin application on all of the POX isozymes, total enzyme activity was measured (Figure 2.3). At week 1, total peroxidase activity was higher in all VAM roots even when % colonization was low. At 2 weeks, +VAM-FOR roots had the highest total peroxidase activity, but +VAM+FOR roots had the lowest activity compared to all other roots (P<0.05). The maximal difference in POX activity at week 2 corresponded to a large variation in colonization by Glomus intraradices between +VAM+FOR and +VAM-FOR roots (Table 2.1). By week 3, POX activity was highest in the control (NAM-FOR) and was lowest in the two mycorrhizal treatments (+VAM-FOR and +VAM+FOR) possibly indicating increased suppression Of POX activity in VAM roots. 160 + -VAM+FOR + +VAM-FOR 140 .1 —A— +VAM+FOR 5‘ 120-1 1 39 as o: 38 25:5 100 — 233 0v 0. 80 J \ 60 - l T l 1 2 3 Weeks after germination Figure 2.3. Total peroxidase (POX) activity in com roots followed by VAM fungal inoculation and formononetin appication. The 100% POX activity for week 1, 2, and 3 ranges between 0.1910 025 0D,, 3" mg" protein. Plants that received Glomus rhtraradrbes and/or an application Of formononetin are indicated as VAM and FOR, respectively ('-' indicates absence and "-1-“ presence). Each point is memeanoffivereplicatesNenicalfinesconespondtothestarIdardenor. 41 DISCUSSION Increased metabolic activity in infected roots, along with the production of fungal biomass and respiration by the endophyte, must be a cost to the mycorrhizal plant (Smith and Gianinazzi-Pearson, 1988). Therefore, enhanced metabolic activity is expected upon mycorrhizal colonization. In our study, increased activity of two malate dehydrogenase isoenzymes (MDH3, MDH4) was Observed in 3-week-old VAM roots compared to controls, and the expression Of these isozymes were further increased in the presence of formononetin. Increased expression of these two Isoenzymes is probably linked to the amount Of fungal biomass (correlated with % colonization in G. intraradices). This expression is likely related to the additional energy required by both symbionts to generate new cellular structures. Fungal biomass was not separated from the root tissue, and therefore, we cannot prove that the two differently expressed MDH isozymes are plant-derived. It is possible that the marked increase in MDH4 was actually due to fungal enzymes. Rosendahl (1992) found similar, unique MDH isofonns, and suggested that these mycorrhizae-specific MDH isozymes were synthesized by the AM fungus. The fact that P-sufficient mycorrhizal plants are likely to be more efficient at carbon utilization (Snellgrove et al., 1982) suggests that catabolic enzymes must work at a higher level of activity compared to non-VAM plants. It should be noted that in our study there were no differences In root protein content (data not shown) and dry weight. Therefore, the increased isozyme activity was directly related to higher metabolic rates in these mycorrhizae. Regardless of its source, MDH4 appeared to be strongly expressed in VAM associations in maize 42 and could serve as a specific, early indicator for Z. mays-G. intraradices colonization. Hepper at al. (1986) used native PAGE to identify AM fungi in leek (Allium porum L) and maize (Z. mays L) roots based on the presence Of certain esterases. In our results, a marked increase in EST activity, especially in EST5, was noted at 2 weeks of growth, when there was rapid fungal colonization and fcrrnononetin enhanced fungal spread. Our data indicates that if fungal esterases were present, they were not well resolved from maize isoenzymes, and the fungal EST made only a small contribution to total esterase activity. More likely, colonization of the roots by a VAM fungus stimulates the host plant to respond by increasing the level of non-specific catabolic esterases, such as with EST 1, 2, 3, and 5 activity compared to nonmycorrhizal roots. In addition, these EST activities were further increased in the presence Of fcrrnononetin. Increased EST activities did not correspond in time with increased MDH, suggesting that alterations in EST were not correlated with fungal biomass, but may have been triggered by fungal elicitors since the increased EST activity preceeded shifts in MDH activity. Alterations of EST isozymes is yet another indication Of the enhanced metabolic activity during the establishment of AM associations, especially in the presence of fcrrnononetin. To determine whether a host plant responds to the endophyte in a manner similar to that of a pathogen, peroxidase, an enzyme associated with host defense, was monitored. In leek, both Chitinase and cell-well-bound peroxidase have increased above the non-VAM levels during the first few days of VAM colonization 43 but then decreased below those of non-VAM roots at later stages of the symbiosis (Spanu and Bonfante-Fesolo, 1988; Spanu et al., 1989). This suggests that VAM infection initially elicits a weak defense reactions by the host (Gianinazzi-Pearson, 1991) but later there is suppression of the host response. In the presence Of VAM fungi, total POX activity was found to be higher only at the first and second week Of growth. Although small amounts of fungal biomass were present in these roots, the host response to this very early stage Of colonization possibly indicate the Change Of fungal saprophytic state to infective state (Giovannetti et al., 1994). By 3 weeks of growth, POX activity of mycorrhizae was less to that Of control roots, in agreement with Spanu and Bonfante-Fasolo (1988). Apparently recognition between host and endophyte had already occurred, and the host was actively suppressing this defense response at a very early stage Of the symbiosis. A dramatic effect of formononetin application was seen at week 2, where +VAM+FOR plants treated with this isoflavonoid had the lowest POX activity. This reduction in total POX activity may be a response of the host, concomitant with rapid fungal colonization in young corn roots (T able 2.1), since the reduction in POX coincided with the most rapid spread Of the fungus. It is interesting that even at week 1, when infection is barely detectable, peroxidase activity in mycorrhizae-roots is significantly elevated over non-VAM roots. Such an alteration Of the host’s defense response is reminescent Of VOIpin at al. (1994) where the mere presence Of VAM spores and hyphae in the rhizosphere was enough to increase Chitinase and Chalcone isomerase activities. The decrease in total peroxidase activity triggered by fcrrnononetin application in +VAM+FOR roots 44 compared to +VAM-FOR roots at weeks 2 and 3, and in -VAM +FOR roots compared to NAM-FOR roots at week 3, was not expected. This finding suggests a possible mechanism for the effect that fcrrnononetin has on colonization by VAM fungi. Increased activity of the cell wall-bound anionic peroxidases will increase the cross links between cell wall polysaccharides, as well as hydroxyproline-rich glycoproteins, thus making the cell well more rigid and more resistant to rapid fungal spread. The contact between a mycorrhizal fungus and the host, or even the presence of such a fungus in the rhizosphere, will result in an increase in peroxidase activity due to an early defense response being elicited in the plant. When fcrrnononetin is present during the early contact and penetration phases of VAM colonization, the cell well does not become as rigid due to the decreased activity Of the cell wall peroxidases, and fungal spread is rapid and facile. Therefore, it appears that the effect of fcrrnononetin on peroxidases occurs only in the presence of the fungus, since in non-VAM roots application of fcrrnononetin alone did not cause a decrease in POX activity at week 1 and 2 (Figure 2.3). Since fcrrnononetin production is involved with stress response, perhaps high levels Of this isoflavonoid in the presence Of the VAM fungus may be essential in triggering the suppression response. In conclusion, this study has pointed out the potential value of monitoring specific isozymes during the establishment Of mycorrhizal associations. If a larger number Of isozyme activities were examined, a more complete picture of root metabolism and VAM fungal physiology could be obtained. Consistent differences in MDH and EST isozymes following mycorrhizal colonization may be used as 45 parameters to evaluate active infections of VAM fungi at early stages of host- endophyte interactions. At later stages of mycorrhizal symbioses, other enzyme activities such as Mg'ATPases, phosphatases or auxin oxidases might give new insights into the functioning of mature Glomus associations. Our results also suggest that high levels of certain secondary metabolites may assist in the host suppression Of certain hydrolytic enzymes. Discovery of new molecular and physiological markers that define a mycorrhizal symbioses in terms of new biochemical capabilities is essential to understand the interplay between plant and microbe. The effect that P nutritional status has on these symbioses and on the physiological alterations reported here is currently underway in this laboratory. LIST OF REFERENCES Bonfante-Fasolo P. and Grippiolo R. (1982). Ultrastructural and cytochemical Changes in the wall of a vesicular-arbuscular mycorrhizal fungus during symbiosis. Canadian Joumal of Botany 60, 2302-2312. Bonfante-Fasolo P. and Gianinazzi-Pearson V. (1986) Wall and plasmalemma modifications in mycorrhizal symbiosis. In Physiological and Genetical Aspects of Mycorrhizae (V. Gianinazzi- Pearson and S. Gianinazzi, Eds), pp. 67-73. INRA Press, Paris. Bonfante-Fasolo P. (1987) Vesicular-arbuscular mycorrhizae fungus-plant interactions at the cellular level. Symbiosis 3, 249-268. Bradford M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254. Bronner R., Wespthal E. and Dreger F. (1991) Enhanced peroxidase activity with the hypersensitive response Of Solanum dulcamara to the gall mite Aceria cladophthirus (Acari: Eriophyoidea). Canadian Journal of Botany 69, 2192-2195. Davis 8. J. (1964) Disc electrophoresis. ll. Method and application to human serum proteins. Annals of New York Academy of Science 121, 404-427. Dehne H.W. (1986) Influence of VA mycorrhizae on the host plant physiology. In Mycorrhizae: physiology and genetics (V. Gianinazzi and S. Gianinazzi, Ed), pp.431-435. INRA Press, Dijon, France. Dumas E., Gianinazzi-Pearson V. and Gianinazzi S. (1989) Production of new soluble proteins during VA endomycorrhiza formation. Agriculture Ecosystems and Environment 29, 111-114. Gianinazzi-Pearson V. (1991). Protein expression in vesicular-arbuscular endomycorrhizas. In IV Reuniao Brasileira sobre Micorrizas, pp. 33-46. EMBRAPA - UFRRJ, Rio de Janeiro, Brazil. Giovannetti M., Sbrana C. and Logi C. (1994) Early processes involved in host recognition by arbuscular mycorrhizal fungi. New Phytologist 127, 703-709. Hepper C. M., Sen R. and Maskall C. S. (1986) Identification of vesicular- arbuscular mycorrhizal fungi in roots of leak (Allium porrum L.) and maize (Zea mays L) on the basis of enzyme mobility during polyacrylamide gel electrophoresis. New Phytologist 102, 529-539. 47 Jakobsen I. and Rosendahl L (1990) Carbon flow into soil and extemal hyphae from roots Of mycorrhizal cucumber plants. New Phytologist 115, 77-83. Kormanik P. P. and McGrawA C. (1982) Quantification Ofvesicular-arbuscular mycorrhizae in plant roots. In Methods and principles of mycorrhizal research (N.C. Schenck, Ed.), pp. 37-45. Lynn D. G. and Chang M. (1990) Phenolic signals in cohabitation: Implications for plant development Annual Review Plant Physiology and Plant Molecular Biology 41, 497-526. Nair M. G., Safir G. R. and Siqueira J. O. (1991) Isolation and identification of vesicular- arbuscular mycorrhiza-stimulatory compounds from Clover (Trifolium repens) roots. Applied and Environmental Microbilogy 57, 434-439. Pacovsky RS. and Fuller G. (1988) Mineral and lipid composition Of Glycine- Glom us- Bradyrhr'zobium symbioses. Physiologia Plantarum 72, 733-746. Pacovsky R. S. ( 1989a) Carbohydrate, protein and amino acid status of Glycine- Glomus- Bradyrhizobium symbiosis. Physiologia Plantarum 75, 346-354. Pacovsky R.S. (1989b) Metabolic differences in Zea-GIomus-Azospirillum symbioses. Soil Biology and Biochemistry 21, 953-960. Phillips J. M. and Hayrnan D. S. (1970) Improved procedure for clearing roots, and staining parasitic and vesicular- arbuscular mycorrhizal fungi for rapid assessment of infection. Transactions of the British Mycological Society 55, 158- 161. Ridge I. and Osborne D. J. (1970) Hydroxyproline and peroxidases in cell walls of Pisum sativunr regulation by ethylene. Journal of Experimental Botany 21, 843- 856. Rosendehl S. (1992) Influence of three vesicular-arbuscular mycorrhizal fungi (Glomaceae) on the activity Of specific enzymes in the root system of Cucumis sativus L Plant and Soil 144, 219-226. Siqueira J. O., Safir G. R. and Nair M. G. (1991) Stimulation of vesicular- arbuscular mycorrhizae formation by flavonoid compounds. New Phytologist 118, 87-93. Shaw C. R. and Prasad R. (1970) Starch gel electrophoresis. A compilation of recipes. Biochemical Genetics 4, 297-320. Smith S. E and Gianinazzi-Pearson V. (1988) Physiological interactions between symbionts in vesicular-arbuscular mycorrhizal plants. Annual Review of Plant Physiology and Plant Molecular Biology 39, 221 -244 Snellgrove R. C., Splittstosser W. E., Stribley D. P. and Tinker P. B. (1982) The distribution of carbon and the demand of the fungal symbiont in leek plants with vesicular-arbuscular mycorrhizas. New Phytologist 92, 75-87. Spanu P. and Bonfante-Fasolo P. (1988) Cell-well-bound peroxidase activity in roots of mycorrhizal Allium porrum. New Phytologist 109, 119-124. Spanu P., Bonfante—Fasolo P. and Boller T. (1989) Chitinase activity and VA mycorrhiza development. Agriculture, Ecosystems and Environment 29, 309- 313. 49 Volpin H., Elkind Y., Okon Y. and Kapulnik Y. (1994) A vesicular-arbuscular mycorrhizal fungus (Glomus intraradices) induces a defense response in alfalfa roots. Plant Physiology 104: 683-689. Wyss P., Boiler T. and kaen A (1991) Phytoalexin response is elicited by a pathogen (Rhizoctonia soIanr) but not by a mycorrhizal fungus (Glomus mosseae) in soybean roots. Expen'entia 47: 395-399. CHAPTER 3 PHOSPHORUS EFFECT ON ISOENZYME EXPRESSION IN ENDOMYCORRHIZAL MAIZE INTRODUCTION Phosphorus is an essential nutrient for all cells. When organisms absorb their mineral nutrients directly from the extemal medium, orthophosphate (Pi, H2P0; or HPOf) is the form Of P that is preferentially assimilated. Based on its contribution to biomass, Pi is one of the least available mineral nutrients in many environments (Marschner, 1986), mainly due to low soil P concentration (micromolar range) and the slow diffusion of Pa in the soil. The presence of vesicular-arbuscular mycorrhizal (VAM) fungi in the roots of plants enhances P uptake under deficient or moderate available soil P. Mycorrhizal fungi have considerable ability to absorb and translocate nutrients, especially P (Cooper and Tinker, 1978), and the distances over which translocation can take place exceeds the depletion zone likely to develop around an actively absorbing root (Rhodes and Gerdemann, 1975). Mycorrhizal roots are able to use organic forms of P (Jayachandran et al., 1992) and absorb sparingly soluble P in high P-fixing soils (Gianinazzi-Pearson et al., 1981), whereas non-mycorrhizal roots depend almost exclusively on Pi absorbed from the external solution for P nutrition (Marschner, 1986). 51 Although direct fungal activity in mycorrhizal roots is exceedingly difficult to distinguish either from enhanced root-surface or rhizosphere activities (Smith and Gianinazzi-Pearson, 1988), studies on enzyme activities may give new insights into this process. Acid phosphatases (ACP) release inorganic phosphate from organic phosphate compounds (Suticliffe and Sexton, 1968), and the activity of ACP enzymes are generally increased in roots growing under P stress (Woolhouse, 1969). Intracellular and extracellular ACP are integral components of the plant response to Pi deficiency (Goldstein et al., 1988), which is a common occurrence. Therefore, the regulation Of these enzymes is critical to a plant’s survival (Duff et al., 1991). The association between the plants and VAM fungi is favoured under low P availability (Schwab et al., 1991). Thus, as aquatic plants began the evolutionary process of becoming terrestrial, they became colonized by VAM fungi as a means of coping with P deficiency. Among various enzyme systems involved in P metabolism, acid phosphatase (ACP) and alkaline phosphatase (ALP) may play an important role in mycorrhizal symbiosis. In G. mosseae. ACP activity significantly increase in vacuolated terminal hyphae of the arbuscule branches, particularly in the dense cytoplasm, the mitochondria, in small vacuoles along the tonoplast and within the walls Of intercellular hyphae (Gianinazzi et al., 1979). Similarly, ACP activity in mycorrhizal been roots was significantly greater than non-VAM roots (Pacovsky et al., 1991). Furthermore, VAM fungal vacuoles are Characterized by the presence of an ALP (Gianinazzi et al., 1979; Tisserent et al., 1993), and the activity of this enzyme is related to active infection, well-developed arbuscules (Gianinazzi- Pearson and Gianinazzi, 1978), and stimulation of plant growth (T Isserant at al., 1993). Therefore, ACP and ALP enzymes may perhaps be involved in the active mechanism of phosphate transport within hyphae of VAM fungi. To quantify the amount of fungal colonization, various techniques have been used. Staining techniques reveal fungal structures in mycorrhizal roots while biomass techniques quantify amounts of fungal material, but neither method gives an indication of the metabolic state of the fungus. Histochemically techniques, using succinate dehydrogenase staining, can detect the proportion Of metabolically active fungal mycelium (Smith and Gianinazzi-Pearson, 1990). Previously, one MDH isozyme was strongly expressed in mycorrhizal corn roots, which was further increased when fcrrnononetin was applied to the soil (Fries et al., 1995). Fonnononetin increased mycorrhizal establishment on corn and clover roots (Fries etal., 1995; Siqueira etal., 1991). Therefore, the cause of increasing MDH isozyme activity remains an unresolved question. Whether the increase in isozyme activity was due to the fungal biomass directly or due to the higher metabolic activity Of host tissue can not be ascertained at this time. Thus, different P status of the soil were used in the present study. Among various enzyme systems, esterase (EST) have been used to identify VAM fungi in roots of leek and com, on the basis of enzyme mobility during polyacrylamide gel electrophoresis (Hepper et al., 1986). They found that the major bands of EST activity in Glomus caledonium or Glomus mosseae had the same mobility as those of corn and so this isozyme was not useful for characterization in this host, although fungal EST isozymes was successfully Identified in leek roots. An increase in EST activity occurred only with rapid infection by G. intraradices in corn. Therefore, studying this enzyme system under different levels of P in the soil could provide a clue regarding root physiology under these conditions. Defense-related enzymes may also change in the presence of mycorrhizal colonization (Spanu et al., 1989; Fries et al.,1995). The ability of the host plants to produce hydrolytic enzymes may modulate the fungal development within the roots. Chitinase and cell-wall-bound peroxidases increase during the first few days of VAM colonization, but then decrease at later stages (Spanu end Bonfante-Fesolo, 1988: Spanu et al., 1989). We found that peroxidases increased at two weeks post- gerrninetion in mycorrhizal corn roots, and subsequently decrease below the levels of control (Fries et al., 1995). In addition, formononetin-treated roots had a different response related to the expression of POX. At two weeks of com growth, fcrrnononetin increased the root colonization and decrease total POX activity compared to mycorrhizal roots alone with one level Of soil P. Increased hyphal growth in the presence of formononetin (Nair et al., 1991) and the effect on root colonization in clover (Siqueira et al., 1991) and corn (Siqueira et al., 1992; Fries et al., 1995) were demonstrated. Since the amount of soil P available to plants influences the development of mycorrhizal symbiosis, it is of importance to study the response of plants, in terms of POX activity, under various P levels in the soil. Higher levels Of soil P can inhibit mycorrhizal colonization, but the mechanisms are not yet known. Various hypothesis have been raised regarding the reduced quantity of root exudates (Graham et al., 1981), exudate quality (Elias and Safir, 1987), or membrane permeability (Ratenayke, 1978) may be one cause for the reduced mycorrhizal colonization under high soil P. There is little information concerning the pattern of ACP, ALP, MDH, EST and POX enzyme activities that can be directly affected by P supply or likely to be involved in the response of plants to mycorrhizal infection under different levels of soil P. Therefore, the purpose of this study was to determine physiological changes that might occur in mycorrhizal plants, growing under different soil P levels. Fonnononetin was exogenously amended to the soil, in order to investigate the physiological changes that may occur with increased colonization level promoted by this isoflavonoid, especifically in corn roots. Acid and alkaline phosphatases (ACP and ALP, respectively), malate dehydrogenase (MDH), esterases (EST), and peroxidases (POX) were the enzymes studied. MATERIALS AND METHODS Experimental design Com plants (Zea mays L cv. Great Lakes-hybrid 582) were grown in soiVsand mixture for 3 weeks. The experimental design consisted of a 5X2X2 factorial with 5 levels of P added to soil (0, 0.25, 0.50, 1.00, and 1.50 mg P mf‘), two levels of VAM fungal inocula (-VAM and +VAM) and two fcrrnononetin applications (-FOR and +FOR) for a total of 20 treatments. The four base treatments consisted of; control (NAM-FOR), either fcrrnononetin (-VAM+FOR) alone or Glomus intraradices Schenck & Smith (+VAM-FOR) alone, or both fcrrnononetin and G. intraradices (+VAM-I-FOR). Each of the four base treatments were fertilized with one 55 of the five P levels. There were five replications per treatment for a total of 100 plants. Growthconditions Three corn seeds were sown in 1.5 kilograms of e steam-sterilized greenhouse top soil/silica sand (1:1, v:v) mix, and plants were thinned to one seedling per pot post-germination to achieve a uniform stand. Soil analysis indicated the mix had a pH Of 7.1, and 117 mg N03, 7.5 mg P, 13.5 mg K, 251 mg Ca, 88 mg Mg, 22 mg Mn, 2 mg Zn, and 1.1 mg Cu per kg soil. Half Of the pots were inoculated with spores of Glomus intraradices INVAM UT 143-2 (purchased from INVAM, Morgentown, West Virginia). Spores were isolated from the soil by wet sieving through 420 mm and 38 mm mesh slaves, and 10 ml of a spore suspension containing 200 spores ml‘ was added to the +VAM substrate, prior to planting. The -VAM pots were left non-inoculated and received 10 ml of a spore suspension filtrate. Then to +FOR pots, 200 ml of a 4 png solution Of fcrrnononetin (Rhizotech lnC., Hopewell, NJ) was added to the soil at onset of the experiment to complete the 5 X 2 X 2 factorial design. Plants were grown in a growth chamber with 300 pmol m4 s" of photosynthetic active light with a 14 h day length, and 28'C or 21 'C as the day or night temperatures, respectively. Phosphorus amendments consisted of 0 (P-1), 16.6 (P-2), 33.4 (P-3), 66.6 (P-4), and 101.2 (P-5) pg of Pg" Of soil, as K,HPO, and NH,I-l,PO,. Together with the initial soil P level of 8 pg' 9" of soil, this amounts to total P level of 8 (P-1), 24 (P-2), 41 (P-3), 74 (P-4), and 110 (P-5) pg of P g" of soil. N and K were equalized in the soil amendments to 80 pg N g" Of soil (as NH,I-I,PO, and NH,NO,) or 100 pg‘ 9" of soil (as KzHPO, and K280, ). Plants received a basic nutrient solution every other day, beginning 2 days after seedling emergence. The nutrient solution contained 0.25 mM CaCI,, 0.5 mM K280“ 2.5 mM NH,NOs, 1.0 mM (NH,).,SO,, 25 mM H3803, 20 mM FeNaEDTA, 2.0 mM ZnSO;, 0.5 mM CuSO4 and 0.4 mM NazMOO, (modified from Pacovsky and Fuller, 1988). Additional N and K were provided once with the P amendment. Nutrient solution and distilled water were applied to both top and bottom Of each pat (catchment dish present). Growth response and calculation At harvest, corn roots were carefully separated from soil and washed with distilled water. Fresh weights were measured on both shoots and roots. Shoots and a root sub-sample were oven dried at 65’C until a constant weight was Obtained (3 days). Dried roots were then ground in a WIley mill and 30 mg was subjected to digestion with H280, for a phosphate detenninetion, as previously described (Nelsen and Safir, 1982). The remainder of the roots were wrapped in aluminium foil, immediately frozen in liquid nitrogen, and stored at -80'C until use. A portion of the root sample was thawed, cleared and stained with trypan blue (Phillips and Hayman, 1970), and percentage of root segments colonized by G. intraradices was estimated by the line-intersect method (Korrnanik and McGraw, 1982). Frozen roots were then placed in a cooled mortar containing liquid N2 and were ground with pestle in the presence of liquid N2 until finely powdered. martial Pulvt containning mill dithio‘ fluoride, ar homogenlz mtr’iugal Chicago, ll titles at . determine standard, I “9th am POIyacryl Se “mph acelilting phosphate Derollltlats DiocedUr E Total acir Me Preparation of root extracts Pulverized root tissue was mixed with grinding buffer (0.75 ml 9" fresh root) containning 50 mM Tris-HCI, pH 7.0, 3.0 mM ethylenediamine tetraecetic acid, 2.5 mM dithiolthreitol, 250 mM sucrose, 50 mM NaCl, 2mM phenylmethylsulfonyl fluoride, and 2 mM N-ethyI-maleimide (Pacovsky, 1989). Cooled samples were then homogenized with a tissue grinder (T ekmar, Cincinnati, OH), followed by centrifugation for 10 min at 12,000 x g at 4'0 (Marathon 21K/BR, Fisher Scientific, Chicago, IL). The supemetants were collected and stored frozen in microcentrifuge tubes at -80'C until use. Bradford's (1976) dye-binding assay was used to determine the protein concentration in each sample using bovine y-globulin as a standard, and root protein content was calculated using root moisture content, fresh weight and extract volume for each sample. Polyacrylamide native gel elechopharesis Samples were then prepared for discontinuous native polyacrylamide gel electrophoresis (PAGE) using a 4 % stacking gel and a 7.5 % separating gel according to Davis (1964). Thirty pg of total root protein were loaded per well. Acid phosphatase (ACP), malate dehydrogenase (MDH), esterase (EST), and peroxidase (POX) isoenzymes were visualized using standard activity-staining procedures (Shaw and Prasad, 1970). Total acid and alkaline phosphatase measurements Measurements of total acid phosphatase activity was determined spectrophotometrically by measuring the amount of p-nitrophenol released by 10 pg of protein from p-nitrophenylphosphate. The quantitative assay for acid phosphatase was modified from a colorimetric technique (McLachlan, 1982) using a 02 M sodium acetate-acetic acid buffer at pH 5.0 with 12.5 mg p- nitrophenylphosphate per 25 ml Of buffer. Ten pg of protein was added to 5 ml of substrate and was incubated in the dark at 30' C for 30 minutes. The reaction was terminated by adding of 1 ml of reaction mixture to 4 ml of 2 M NeOH. Optical density Of the p-nitrophenol released was measured at 405 nm using a Perkin-Elmer Model 35 spectrophotometer. The Standard curve was performed as described by Tabatebai and Bremner (1969). Acid and phosphatase activity was expressed in terms of pg Pi released ‘ min" mg" protein. Total alkaline phosphatase were determined spectrophotometrically following the same procedure as for acid phosphatase, except that the substrate used was 0.05 _M_ iris-citric acid, pH 8.5. Alkaline phosphatase activity was expressed in terms of pg Pi released min"' mg" protein. Total peroxidase activity measurements Measurements of total peroxidase activity were determined spectrophotometrically following a modified version of Ridge and Osborne (1970). Ten pg of protein from each sample was added to 4 ml of 11.36 mM H20, in 8 mM phosphate buffer, pH 7.0, and 1 ml of 1.96 mM guaiacol. Optical density at 480 nm was recorded every 30 seconds for 10 minutes in a Perkin-Elmer Model 35 spectrophotometer. Five replicates from each treatment were assayed. Total peroxidase activity was expressed as 0D,, s"mg" protein. Statistical analysis The entire experiment (2 X 2 X 5 factorial with 5 replications per treatment) was repeated twice, and the results for plant growth, phosphorus concentration, colonization, peroxidase, acid phosphatase, alkaline phosphatase assays and isoenzyme pattems were similar. Dry weight, phosphorus concentration, and root protein content date, from one experiment, were subjected to a complete analysis of variance. Treatment means were compared by least significant difference (LSD) test (P<0.05), while colonization means were compared by Student’s t-test (P < 0.05). RESULTS Growth Shoot dry weights of +VAM-FOR and +VAM+FOR plants were greater than NAM-FOR plants at the lower soil P application rates (Table 3.1). At higher soil P applications, there were no Significant difference between the treatments. In contrast, root dry weights differed only at higher levels. At P-4, mycorrhizal roots weighed less than controls, whereas at P-S mycorrhizal roots alone (+VAM-FOR) weighed significantly less than the control (NAM-FOR) roots. The positive effect of AM colonization on phosphorus uptake was observed at limiting levels of P (P-1 and Table 3.1. Growth, root phosphorus and root protein concentration of corn plants Inoculated wlth Glomus intraradices (+VAM) and fcrrnononetin (+FOR) as affected by different levels of phosphorus applied In the sell. Plants were harvested at 3 weeks of growth. Treatments Total Soil Shoot Root Root Root Phosphorus Dry Dry Phosphorus Protein Weight Weight Concentration Concentration (vs '9") (e) (a) meme") (me '9") - VAM -FOR 8 0.67 b 0.53 a 1.92 C 2.62 b - VAM+FOR 8 0.79 a 0.53 a 2.01 b 3.00 b +VAM- FOR 8 0.74 ab 0.46 a 2.28 e 3.90 a +VAM+FOR 8 0.79 a 0.54 a 2.24 a 4.54 a - VAM -FOR 24 0.88 b 0.72 a 2.25 b 2.69 b - VAM+FOR 24 1.16 a 0.79 e 2.10 b 2.73 b +VAM- FOR 24 1.20 a 0.80 a 2.72 a 3.96 a +VAM+FOR 24 1.22 a 0.74 e 2.84 a 3.27 ab - VAM -FOR 41 1.15 a 0.74 e 2.67 be 3.75 e - VAM+FOR 41 1.19 a 0.76 a 2.53 C 3.22 a +VAM- FOR 41 1.23 a 0.71 a 2.78 ab 3.39 a +VAM+FOR 41 1.30 a 0.68 a 2.89 e 3.20 e - VAM -FOR 74 1.18 a 0.77 a 3.36 a 3.27 e - VAM+FOR 74 1.27 a 0.75 ab 3.01 b 3.70 a +VAM- FOR 74 1.33 a 0.62 c 3.25 ab 3.17 a +VAM+FOR 74 1.34 a 0.64 bc 3.53 a 3.21 a - VAM -FOR 110 1.19 a 0.73 a 5.31 a 2.50 a - VAM-I-FOR 110 1.27 a 0.68 ab 4.85 a 2.69 a +VAM- FOR 110 1.30 a 0.58 b 4.80 a 2.55 a +VAM+FOR 110 1.35 a 0.72 ab 5.33 a 2.01 a Means (5 replicates per treatment) followed by the same letter within each P treatment did not differ significantly by Least Significant Difference test (P< 0.05). 61 P-2; Table 3.1). In the presence of fcrrnononetin, the increase in root phosphorus concentration was significantly different from controls at P-1, P-2, and P3 At higher soil P concentration, mycorrhizal roots did not differ significantly from the control roots. Protein content of mycorrhizal roots was higher than controls at the lower P levels (P-1 and P-2). Fonnononetin-treated plants did not differ significantly from mycorrhizal roots (+VAM-FOR), and controls at P-2. Calculation Colonization by G. intraradices significantly increased in the presence of fcrrnononetin at P-1, P-2, and P-3 (Figure 3.1). The difference was 11, 23 and 10.8% greater for +VAM+FOR plants, respectively, compared to +VAM-FOR plants. Colonization of mycorrhizal roots significantly decreased when total soil P was 24 pg‘g" of soil (P-2) , whereas increasing P in the soil decreased colonization in a steady manner (Figure 3.1 ). Total acid phosphatase activity There was a pronounced decrease in total acid phosphatase (ACP) activity as P applied increased (Figure 3.2). At the lowest P levels (P-1, P-2, and P-3) mycorrhizal roots had significantly higher ACP activities than did non-VAM (approximately 23 %), but at the higher P levels (P-4, PS) the difference in ACP activity between VAM and non-VAM plants was insignificant ( less than 4 %). If a linear regression Is applied to ACP data from all treatments versus total soil P, the r1 55 O +VAM- FOR 0 +VAM+FOR 3S- Colonlzatlon ( % ) 25-I 2° I I I I I Total P In the soil (pg P '9") Figure 3.1. Rootcolonizationfromthreewwk-oldmyaorrhizal(Glomus intraradices) Zea mays roots grownunder5dfferentsoilPlevels(P—1= 8; P-2=24; P-3=41;P-4=74andP-5=110pgg"ofsofl) hthepresenceorebsenceofexogenouslyappliedformononetin. Plartsthatreceivedtheappbatian afformononetinereindicatedes-I-FOR. Eachpoirtisthemeanoffiverepicates.Verticalines correspond to the standard error. +VAM-FOR (y: 43.9 - 0.16x; r=-0.83); +VAM+FOR (y= 50.6 - 020x; r='0.88). A 22 .5 P4 «g + - VAM - FOR E 1 —o— - VAIII «1 FOR '- 20 -‘ b -e— + VAM - FOR _E ‘ —<>— + VAN + FOR '.E .E 18 -i E 8 - P-2 2 16 d O. P. \ P4 at 3 '. 3? 14 -1 .2 ‘6 ° 1 III 3 P4 8 12 — \ a . \ a e\ P" 8 \e 2 e o < 3 I l l T l 0 20 40 60 80 100 120 Total P In the soil (pg P'g" soil) Figure 3.2. Total acid phosphatase activity (ACP) from three week-Old mycorrhizal and non- mycorrhizaIZea mays rootsgrown underSdil‘ferent soil P levels (P-1= 8; P-2 = 24; P-3 =41; P-4=74andP-5=110pg.g"ofsoil)inthepresenceorabsenceofexogenouslyapplied fonmmnetm.ThetotelACPactMyisemressedasngireleased'mm"mg"ofprotein. Plants that received Glomus intraradices and/or an application of formononetin are indicated as VAM andFOR, respectively ('-'hdicatesabsenceend'+'presence).EachpoiI'Itisthemeanoffive replicates. Vertical fines correspondto the standard error. slope indicates that 65 ng Pi min" mg" of ACP enzyme activity is last for each additional microgram of P applied to the soil (y = -0.065 x + 16.34; t’: 0.88). Total allallne phosphatase activity Alkaline phosphatase (ALP) activity (Figure 3.3) followed a similar trend as was seen for acid phosphatase (Figure 3.2). In general, mycorrhizal roots had ALP activities that were 7.7 % to 3.6 % greater than non-VAM roots (not statistically significant). As applied phosphate increased, the decrease in ALP activity (40ng P, released min" mg" ; y = -0.04 x + 14.85; I’: 0.90) was 38 % less than that for ACP activity indicating that ALP was not as responsive to P nutrition as ACP. Interestingly, the presence of VAM fungi was not capable of enhancing ALP activities either. Acid Phosphanse Isazymes Nine acid phosphatase isozymes were observed in activity-stained gels for both VAM and non-VAM roots (Figure 3.4). There was a general decrease of acid phosphatase (ACP) activity with the increase of P levels in non-mycorrhizal roots (ACP1, 2, 6, 7, 8, 9). All ACP isozymes showed an increase in VAM roots at the lower P levels (P-1, P-2, and P-3) compared with non-VAM roots. The ACP activity of +VAM+FOR roots at P-1 was higher than any other treatment. The +VAM+FOR treated roots showed highest ACP activity even at 74 pg of P per g of soil (Figure 3.4, P-4), but then this activity decreased at higher P levels. E 22 3 g —e— - VAM- FOR .. —o— - VAM+FOR 3 2° “ —e— +VAM- FOR .5: —<>— +VAM+ FOR '5 =3 18 4 O 8 P-1 2 --I a— 16 at 5 .E‘ > 14 — § 5 .2 _ a Q o '5. 8 10 — 2 3 I l I l I o 20 40 so so 100 120 Total P In the soil (pg P’g" of soil) Figure 3.3. Total allaline phosphatase activity (ALP) from three week-old mycorrhizal and non- mycorrhizalZeamaysrootsgownmderSdlI'lererItsoilPlevels(P-1=8;P-2=24;P-3=41;P-4=74 andP-5=110pg.g"dsol)hthepresaoeorebsuoedexogamslyappfiedfomomnahm totalALPectiviyisemressedesngireleasedmh"mg"ofprotein.PlartstereceivedGlamus r’ntraradr’cesandloran applicationof formononetin are indicated as VAMand FOR, respectively C—‘itdcatesabsertceertd'+'preserlce).Eachpoittisthemeanoffivereplicates.Verticalbtes correspondtothestandarderror. Figure 3.4. Banding pattern for acid phosphatase (ACP) isoenzymes visualized it e 7.5 % polyacrylamide native gel. Protein extracts (30 pgllane) fromthree-week-old mycorrhizaland non— mycorrhizalZeamays rootsgrownurtder5differentsoilPlevels(P-1 =8; P2 = 24;P-3=41; P-4=74andP-5=110pg.g"ofsoil)inthepresenceorabsenceofexogenouslyappied formononetinPlarItsthatreceived Glornusintraradicesandloranappicetionofformononetitare indicated as VAM and FOR, respectively ('-" indicates absence and '+" presence). 67 ACID PHOSPHATASE PSI/559‘? 5‘0"???) 3‘9“???) 5‘9"???) 44444 44444 44444 44444 RM ' 0.50 0.58 ACP9 - VAM - FOR - VAM + FOR + VAM - FOR + VAM + FOR MAD-malate dehydrogenase Isozymes Nine isozymes bands were detected in the NAB-dependent malate dehydrogenase (MDH) activity stained gels (Figure 3.5). Among those, one malate dehydrogenase isoenzyme (MDH3) demonstrated higher activity in VAM-colonized corn roots, regardless of the P level applied in the soil (Figure 3.5, +VAM-FOR and +VAM+FOR). A second isozyme, MDH2 was expressed strongly in fcrrnononetin-treated roots, and in mycorrhizae commred to controls. The expression of this isoenzyme was only weakly present in control roots. Forrnononetin-treated plants displayed a weak activity of this isozyme at the lowest P level, increasing in activity as P level increased. In mycorrhizal roots treated with fcrrnononetin, an increase in activity was Observed in low P levels( P-1,P-2 and P-3). Esterase Isazymes Seven bands in the esterase (EST) activity-stained gels were observed (Figure 3.6). There were not obvious differences in the isozyme patterns between the four base treatments. At P-5, all treatments showed a stranger expression of EST1, 2, 3, 4. E517 also was strongly expressed in all treatments at P-5 compared to the other P levels. Total perOIddase actlvlty At the lowest level of P application (P-1, P-2, and P-3), peroxidase activity for mycorrhizal plants was always lower than non-VAM plants (Figure 3.7), by approximately 16 %. At the highest rate of P application, when there was active Figure 3.5. Banding pattern for NAB-malate dehydrogenase (MDH) isoenzymes visuafized 'n a 7.5 °/. polyacrylamide native gel. Protein extracts (so pgllane) from three-week-old mycorrhizal andnon-myconhizalZeamays rootsgrownunderSd‘tferentsoil P levels (P-1= 8; P-2=24; P-3=41;P-4=74andP-5=110pg.g"ofsoil)inthepresenceorebsenceofexogenously applied formononetin. Plants that received Glomus intraradices and/or an application of fcrrnononetin are indicated as VAM and FOR, respectively (9 indicates absence and '+' presence). 70 U h 9 m0u+2<>+ .d .6 .d .d b 8 a l mOm-E<>+ m0u+§<>- . _ 3 e e .e .e .e .e . a». ear 0. eeaeaaaq av mmzmo FEES). _. mOu-E<>- 71 Figure 3.6. Bandingpattern for esterase(EST)isoenzymesvisr.Ializedina7.5%polyacrylamide native gel. Protein extracts (30 pgllane) from three-week-old mycorrhiml and non-mycorrhizal Zearnays rootsgrownunderSrflferentsoilPlevels(P-1=8;P-2=24;P-3=41;P-4=74end P-5=110pg.g"ofsofl)hthepresenceorebsencedexogemmlyappfiedfomomneth. Plants that received Glomus intraradices and/or an application of fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and "+' presence). 72 ESTERASE 73 0.22 —e— - VAN - FOR 020 '7 —o— - VAN + FOR + +VAM - FOR 0.18 _ -<>- +VAM 4» FOR Peroxidase actIVIty (oam°e"-mg" protein) 1/ 0.14 - 0.12 - ‘ ’ O 0.10 - t O 0.08 - )V ° 00 I I I I 1 T 0 20 40 60 so 100 Total P In the soil (pg P g") 120 Figure 3. 7. Total peroxidase (POX) activity from three week-Old mycorrhizal and non-mycorrhizal Zea mays rootsgrownmderSdifferentsoilPlevels(P—1=8; P-2=24; P-3=41; P-4=74andP-5=110 pg.g"dsai)hthepresaoeorabsemedexogmlyappfiedfmmneth1hetdel POXactivity iseiqaressedasOD‘s’Hng" protein. Plantsthatreceived Glomusintraradicesandloranapplicationof fcrrnononetin are indicated as VAM and FOR, respectively ('-" indicates absence and '+" presence). EachWBflwmeandfiverepicatesNeflicallhmmnespondtoflwstendaMamr. 74 Figure 3.8. Bending pattern for peroxidase (POX) isoenzymes visualized it a 7.5 % polyacrylamide native gel. Protein extracts (30 pgllane) from three-week-old mycorrhizal and non- mycorrhizalZeamays rootsgrownwderSdilferentsoilPleveb(P-1=8: P-2=24; P-3=41; P-4=74endP-5=110pg.g“ofsoil)hthepresenceorabsenceofexogemuslyappled fcrrnononetin. Plants that received Glomus intraradices and/or an application of formanonet'n are indicated as VAM and FOR, respectively ("-" indicates absence and '+" presence). 75 PEROXIDASE 013 POX6 POX7 POX8 POX9 POX10 m o P m 0 P-4 P-2 P-1 76 inhibition of fungal colonization mycorrhizal roots had total POX levels higher than their non-VAM counterparts. Most interesting was the fact that at any level of P addition within a given VAM status, the presence of fcrrnononetin decreased POX activity (average of 9.2 % for non-VAM roots and 7.8 % for VAM roots). In general, total POX activity was observed to increase with increasing soil P availability. Peraiddase Isozymes Eleven peroxidase (POX) isozymes were visualized by native PAGE ( Figure 3.8). VAM inoculation or fcrrnononetin application differentially altered POX isozymes. POX9 and POX10 slightly decreased in VAM roots at the lowest P level (P-1). At P-2, VAM roots showed a strong decrease compared to non-VAM roots. At P-3, there were a slight increase in POX activity in +VAM+FOR roots, but this decreased was less than controls. Increased activities in POX isozymes were observed in all treatments at P-4. At the highest P level (P-5), it appears that there was no difference between the treatments In POX activity expression, but it may be that isozyme activity in the polyacrylamide gels has saturated. In general, POX8, 9 and 1 0 increased as the soil P increased. POX11 showed an increase in the highest P level in mycorrhizal roots. DISCUSSION Physiological and biochemical changes occurred in maize roots inoculated with G. intraradices or treated with fcrrnononetin under different levels of soil P. 77 Changes were shown to be dependent on soil P level, the presence of mycorrhizal fungi, or the presence of fcrrnononetin. Fungal colonization and sporulation by VAM fungi is inhibited at high levels of soil P (Sanders, 1975). In fact , excessively high P levels markedly inhibited mycorrhizal colonization. At low soil P levels, mycorrhizal plants accumulated more shoot, root P and root protein than non-VAM plants. Although some shoot growth differences were found during this early lag phase (while nutrients were accumulating) greater differences would be expected to occur later. Nevertheless, we analysed root enzyme patterns of plants growing with G. intraradices and/or fcrrnononetin at different P levels. We have detected enzyme differences very early in the development of corn growth, even before the onset of rapid nutrient- dependent growth responses. Therefore, it was important that root growth between treatments was similar at this stage (Pacovsky et al., 1986), so observed effects would be due to the mycorrhiza and not an indirect effect of altered growth pattern. Mycorrhizal symbiosis can be dramatically affected by P fertilizer amendment (Hepper, 1983). When P availability increases, the fungus become less important to the host and the potential for uncompensated carbon drain increases (Koide and Schreiner, 1992). Such a growth decrease occurred in corn roots at the highest P level, where mycorrhizal roots accumulated less root mass compared to non-VAM roots. Therefore, the VAM fungal biomass undoubtedly increased the C sink in the roots without a concomitant enhancement of relative P concentration compared to "On-VAM roots. Although total root P increased with applied soil P, mycorrhizal '°°ts accumulated significantly more total root P at the lowest applied P level. 78 Colonization of formononetin-treated VAM plants ( y: 50.6-0.20m l’=0.77) exceeded the colonization of mycorrhizal plants alone (y = 43.9-0.16x; r’: 0.69) in a steady linear fashion indicating that fcrrnononetin could partially overcome the influence of P on reducing VAM colonization. This inhibition of intraradical fungal growth is likely mediated by physiological alterations in roots. Root exudates may be involved (Elias and Safir, 1987; Graham et al., 1981). Root membrane permeability decreases as the soil P availability increases and, consequently, colonization by VAM fungi is reduced (Ratanayake et al., 1978). On the other hand, extensive cross linking of the cell walls in the roots may be one factor in reducing colonization in soils containing high P concentrations (Hepper, 1983). The enhancement of mycorrhizal colonization by application of fcrrnononetin to the soil has been shown before, in clover and corn (Siqueira et al., 1991, Siqueira et al., 1 992; Fries et al., 1995). The effect of formononetin was limited by the number of VAM fungi in the soil and the concentration of the compound applied (Siqueira et al., 1991). We have shown that formononetin-stimulatory effects on root colonization by G. intraradices was greatest at low P levels (P-1, P-2 and P-3; Figure 32) compared to mycorrhizal roots alone (approximately 14 %). At higher P levels. the relative response on colonization due to fcrrnononetin is decreased (7.1 %). The mechanisms underlying the effect of fcrrnononetin on VAM fungal colonization are "0t Vat understood, and to gain an insight into the physiological alterations due to this isoflavone, enzyme patterns were studied. 79 Thus, considering that the primary effect of mycorrhizae is to improve phosphate uptake by the host plant, it is of importance to study enzymes involved in phosphate metabolism. Goldstein et al. (1988) described a phosphate-starvation rescue system in higher plants. One component of the system is the production and excretion of acid phosphatases (ACP) in order to access and solubilize organic P in the environment. However, ACP secretion appears to be regulated in a manner that allows a fine tuning of the response to the extemal Pi concentration (Goldstein er al., 1988). In the present work, corn was grown under different soil P levels and ACP activity decreased as P availability increased in all treatments. Mycorrhizal Phaseolus roots (Pacovsky et al., 1991) and mycorrhizal cowpea roots (Thiagarajan and Ahmad, 1994) had increased ACP activity. In these studies. only one level of soil P was used, and therefore, the alterations in ACP activity in a more mature symbiosis could possibly be an indication of soil P exhaustion following rapid utilization of soil P reserves. The role of ACP enzymes in mycorrhizal associations undoubtedly include absorption and assimilation of P. In the present work, as mineral P application increased ACP activity decreased (between 21 % and 45 % reduced). There was a pronounced negative relationship between ACP a<>tivity and soil P levels ( a decrease of between 50 and 60 ng Pi released min"mg" protein each pg of applied P; r’: 0.90). At higher P levels, ACP activity was similar in both VAM and non-VAM roots. Root colonization was also decreased by the greater P availability in the soil. Therefore, the increased ACP activity in VAM roots Was dePendent not only on fungal biomass, but also due to the level of P availability in the soil. Jayachandran et al. (1992) demonstrated that root colonized by VAM fungi were able to solubilize various forms of amended organic P. Therefore, it may be that the increased ACP activity is an attempt by the mycorrhizal roots to solubilize organic P. Mycorrhizal roots increase P uptake through the hyphae, which have a higher affinity to P in the soil (Cooper and Tinker, 1978). Consequently, VAM roots are able to exhaust available soil P more rapidly than non-mycorrhizal roots. so ACP activity is expected to be higher when colonized plants are growing under low levels of soil P. In addition, higher intracellular ACP may allow for more efficient utilization of P in primary metabolism (Duff et al., 1989). Thus, increased ACP may directly relate to the metabolic status of mycorrhizal fungi, and since fcrrnononetin increased colonization, it likewise caused a concomitant ACP increase as well. Therefore, ACP activity could be involved in the increased uptake of P from the soil in mycorrhizal roots, under low to moderate soil P availability. The influence of P application on alkaline phosphatase (ALP) activity followed that of ACP activity. Highest ALP activitites occurred under low soil P supply, in mycorrhizal roots. Forrnononetin-treated plants further increased the ALP activities. Tisserant et al. (1993) suggested that ALP activity was induced by colonization of host roots and that this enzyme could be linked to the efficiency of arbuscular mYOorrhizal infections in terms of growth improvement. In this work, an increase in intraradical mycelium showing ALP preceeded the mycorrhizal growth response in NW”? POrrum and P. acen‘folia, and was this increase considerably lower in non- r esPOnsive P. sativum, at 4 weeks of growth. Ultracytochemistry confirmed that ALP enzym9 is localized within the phosphate-accumulating vacuoles of the intraradical 81 mycelium of G. intraradices, particularly along the fungal tonoplast (Jacquelinet- Jeanmougin et al., 1987), and suggested that this enzyme is involved in the processes of phosphate transfer to root cells by VAM fungi. ALP activity of AM fungi appears to be induced by the host plant (T isserant et al., 1993) since the fungus must first develop within the root prior the activation of the enzyme. Development of nutrient stress in the plant during early growth may also be associated with induction of enzyme activity (T lsserant et al., 1993), but these authors did not use different level of P to determine if P stress is involved. However, our study has shown that the stimulation of ALP activity was soil-P dependent. Higher activities occurred only at lower soil P, while at higher levels, the ALP activity did not differ between VAM and non-VAM roots. At this time root colonization also decreased. Therefore, this fungal enzyme could provide a marker for analysing the symbiotic efficiency of mycorrhizal infections (T lsserant et al., 1993). and low P availability (also favorable to root colonization) may induce the activity of this enzyme in mycorrhizal roots. ALP seems to be associated with active phosphate assimilation in mycorrhizal roots and the induction will depend on the level of P in the soil. There was an increase in corn growth and yield when fcrrnononetin was amended to a P-fixing soil in the field (Siqueira et al., 1992). Since ALP activity increases just before the plant growth response to the mycorrhizal infection occur (Tisserant et al., 1993), the increase of ALP enzymes in VAM roots treated with fcrrnononetin indicates the potential for enhanced corn growth and subsequently greater yield. Thus, the improvement of growth and P uptake in mycorrhizal plants is due to their ability to “mine” the soil. In the symbiosis, the enzymes involved in P metabolism, especially ACP and ALP, must respond to the new method by which P is absorbed, assimilated and transferred. Higher activities for the ACP and ALP is likely to be advantageous for this symbiosis in order to release Pi from the soil (more likely ACP) or in the vacuoles of the fungi to facilitate the transfer of P (ALP). Therefore, the direct involvement of the VAM fungus in alleviating the increased P demand by the host and the enhancing root P concentration is shown by the increased activity of ACP and ALP, under low to moderate levels of P in the soil. Among important physiological alterations that occur in myconhizal roots, enzymes that are linked to P metabolism, are important physiological markers that are involved in a successful mycorrhizal symbioses. ACP activity showed to be more sensitive to the levels of available soil P. Certain dehydrogenases have been used as markers for mycorrhizal symbioses. Succinate dehydrogenase (SDH) has been used histochemically to detect the proportion of viable hyphae in infected roots (Ocampo and Barea, 1985; Smith and Gianinazzi-Pearson, 1990), and it has been used as a physiological marker for the evaluation of active root colonization. In an earlier study (Fries et al., 1 995). mycorrhizal roots significantly increased expression of a malate dehydrogenase (MDH) isozyme in a com-G. intraradices symbiosis. Forrnononetin- treated mycorrhizal roots further increased the expression of this isozyme. Increased fungal biomass could be one factor for enhanced enzyme activity since mycorrhizal roots treated with fcrrnononetin had higher % colonization than mycorrhizal roots without a formononetin application. In this study, MDH3 isozyme was expressed only in mycorrhizal roots, M'lether treated or not with fcrrnononetin. Despite that, mycorrhizal roots accumulated more P and protein at lower soil P, and the difference in MDH isozyme also occurred when there was no significant difference between root P and protein concentration behlveen mycorrhizal and non- mycorrhizal roots. Therefore, we propose that the MDH isozyme expressed in myconhizal roots may be of fungal origin. MDH was also effective as an indicator of the amount of fungal biomass in the roots, and active infection, as for SDH. Furthermore, MDH expression in mycorrhizal com roots was consistent with the amount of fungal biomass in the roots, suggesting that these MDH isozymes can serve as a specific, early indicator of the presence of G. intraradices in corn roots. Esterase isoenzymes were more difficult to analyse in the Zea-Glomus slfl'flbiosis. As seen before (Fries et al., 1995), mycorrhizal roots showed a pronounced effect on EST activity when rapid colonization of roots occurred (2 weeks), whereas at 3 weeks no difference could be detected. Also, identification Of fungal EST isozymes, in polyacrylamide gel electrophoresis, using a mature symbiosis of corn roots colonized by either G. caledonium or G. mosseae was not possible (Hepper et al., 1986). In contrast, they identified different EST isozyme bands that were characteristic of both of these fungi grown in leek roots. Therefore, it seems that analysis of this enzyme system depends on whether the mobilities and Strengths of the host EST isozymes obscure the fungal isozymes. In addition, different soil P levels had little or no effect on the expression of these isozymes. For mycorrhizal corn roots, there may be some physiological importance for this enzyme system only at the early stages of mycorrhizal formation (Fries er al., 1995). There have been recent reports indicating a relationship between peroxidase activity and plant P status, but prior to this paper, practically nothing has been done to characterize the relationship between peroxidase activity and plant-derived phenolics, such as fcrrnononetin. Plant phytohorrnones regulate the levels of peroxidase activity in plants, and their levels decrease as plant growth develops (Ridge and Osbome, 1970). In pathogenic interactions, ethylene production is involved in plant defense response (Ecker and Davis, 1987). Increased ethylene production after pathogen infection can stimulate phenylalanine ammonia-lyase ( PAL) and extracellular POX activity leading to lignification of cell walls. Enhanced ethylene production and extracellular POX activities in mycorrhizal potato roots was observed with increasing abiotic P supply, which causes P-induced decrease in VAM infection (McArthur and Knowles, 1992). In the present study, corn growing with elevated levels of applied P had decreased fungal colonization, while POX activity in the roots increased. In a preWous work, POX activity for mycorrhizal corn roots was lower than controls (Fries et al., 1995). As the P concentration in the soil increases, POX activity also inereases in a linear fashion for all treatments. In lower soil P, POX activity in r1‘3'<>orrhizal roots was below than the controls. Furthermore, fcrrnononetin-treated D'ants decreased the activity of POX enzymes compared to mycorrhizal alone. This may suggest that fcrrnononetin facilitates root colonization by G. intraradices by ‘O‘Nering the POX activity in plant roots. Under high P concentrations, the relatively higher levels of POX activity in mycorrhizal roots might contribute to the restricted 'lltraradical fungal growth. However, POX activity alone is not sufficient to explain the effect of P on the root colonization process in mycorrhizal corn roots or the effect of fcrrnononetin in stimulating root colonization. Nevertheless, this data provide an additional reason why there is a greater potential for resistance to VAM infection under high soil P availability. Plants growing at low P levels were colonized with VAM fungi readily while POX activities were low. At higher soil P availability, plants may limit VAM fungi by increasing POX activity, as a means to slow fungal spread or localize the invasion of the fungi. Peroxidases have an important role in secondary wall formation and suberization, which contribute to the plant’s resistance to invasion by microorganisms (Gianinazzi, 1991). The mechanism of action of formononetin in im proving VAM root colonization may be to reduce POX activities which could be Che way to facilitate the spread of the fungus. These findings suggest that the presence of VAM infection alters the physiology of Z. mays roots, and the host response is closely related to soil available P. In add ition, specific physiological processes (ACP, ALP) were stimulated by low abiotic :3 levels in the soil, and may play a role in the P metabolism. The MDH isozyme a(=‘t'ivity may possibly be considered as a physiological marker to monitor the activity 0* the VAM colonization in corn roots. EST isozymes should not be considered as physiological markers for established Zea mays-G. intraradices symbiosis. POX a‘12tivities were decreased under conditions of low soil P. In higher soil P levels, POX activities of mycorrhizal corn roots were enhanced, possibly accounting for the lower root colonization. Forrnononetin significantly increased the levels of corn root colonization at low soil P availability. Forrnononetin-treated mycorrhizal plants seem to increase the effect caused by mycorrhizal roots alone, when ACP, ALP, MDH enzymes are considered. In addition, the effect of fcrrnononetin was less dependent on the level of P in the soil as compared to mycorrhizal infection alone. Furthermore, fcrrnononetin decreased the root POX activities with a concomitant increase in root colonization of com by G. intraradices. The results presented herein suggested that one way fcrrnononetin acts on rapid root colonization by VAM fungi is by decreasing POX activity, which facilitates the invasion of the fungus in the plant roots. LIST OF REFERENCES Am es R. N., Reid C. P. P., Porter L. and Cambardella C. 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Woolhouse H. W. (1975) Membrane structure and transport problems considered in relation to phosphorus and carbon movements and the regulation of endotrophic mycorrhizal associations. In. Endomycorrhizas (F. E. Sanders, B. Mosse, P. B. Tinker, eds). London, Academic Press, 209-240 inval I melabc lehdue molest alid Cl callabl (All) I mmUlE inClea; these °'°Dpl DElen, ”Ops WOW CHAPTER 4 PLANT GROWTH AND ARBUSCULAR MYCORRHIZAL (Glomus lntraradlces) COLONIZATION AFFECTED BY EXOGENOUSLY APPLIED PHENOLIC COMPOUNDS INTRODUCTION Phenolic compounds are an important class of plant secondary metabolites that are released in the soil as leachates, exudates or by decaying residues (Siqueira et al., 1991a). Several phenolic compounds act as signal molecules or mediate signal transduction pathways in symbiotic systems (Lynn and Chang, 1990). There is increasing evidence that these compounds are capable of influencing the growth and root colonization by arbuscular mycorrhizal (AM) fungi (Siqueira et al., 1991b), and exogenously applied phenolics may stimulate the indigenous population of AM fungi in the field, resulting in increased plant growth and yields (Siqueira et al., 1992). Concentrations of these compounds in the soil are influenced to a great extent by soil type and cropping history (Pederson et al., 1991). Fallow rotations, as well as some perennial or monocropping systems, can either reduce VAM root colonization of crops grown in the following season or can cause negative effects on plant QFOWth (Vivekanandan and Fixen, 1991). Although little mechanistic information is available concerning the negative effects of AM fungi, it has been suggested that allelopathic phenolics may be affecting both the AM fungal diversity as well as their effectiveness in perennial systems, such as in asparagus (Wacker et al., 1990a, 1990b; Pederson et al., 1991) and in monocropped systems (Vivekanandan and Fixen, 1991). Several in vitro studies involving the effects and activity of phenolic compounds on AM fungi have indicated their involvement in the AM symbioses. Depending on the concentration, different flavonoids can either increase or decrease AM fungal spore germination, hyphal growth, and hyphal branching (Gianinazzi etal., 1989; Nair etal., 1991; Tsai et al., 1991; Becard et al., 1992; Baptiste and Siqueira, 1994). The lsoflavones, fcrrnononetin and biochanin A, and to a lesser extent the flavone chrysin have been shown to stimulate root colonization and subsequent plant growth in a Glomus- Trifolium symbiosis (Siqueira et al., 1991b). Other phenolics, such as the phenolic acids, have also been studied. They are commonly found in a variety of soils (Whitehead, 1964; Dalton et al., 1987), have allelopathic activity (Patterson, 1981; Vaughan et al., 1983), and can affect AM fungi. Ferulic acid, an allelochemical commonly found in asparagus roots, decreases root colonization by AM fungi and growth of mycorrhizal asparagus (Walker et al., 1990a). Similarly, diversity of AM fungi and AM fungal spore density in the soil decreases (Walker et al., 1990b). Prior to this report, the effects of other phenolic acids such as p-coumaric and p- hydroxybenzoic acids on the establishment of AM symbiosis have been not stud ied. hydr Plal my: clov Moe on was We have examined the effects of soil applied p-coumaric acid, p- hydroxybenzoic acid and quercetin, with respect to timing and concentration, on AM formation and growth of white clover (Trifolium repens) and sorghum (Sorghum bicolor) plants. MATERIAL AND METHODS Plant, soil and fungal material The effects of several naturally-occuring phenolic compounds on mycorrhizae formation and subsequent plant growth were examined using white clover (Trifolium repens L. cv. Ladino) and sorghum (Sorghum bicolor L. Moench) plants. In all experiments, water-soaked seeds were pre-germinated on filter paper for 24 h and transplanted into plastic pots (4x5.5 cm), each containing 80 g of sterilized sand-soil mix (2:1). A 9.0 cm diameter Petri dish was used under each pot to avoid chemical cross-contamination and to facilitate watering. The soil mix had a pH of 7.1, CEC of 4 meq'100 9“ soil; 11.5 pg P. 9 HQ K. 720 pg Ca, 47.3 pg Mg, 7 pg Zn and 34 pg 9" of soil. (Michigan State University Soil Testing Laboratory), and was inoculated with propagules of Glomus intraradices Schenck and Smith, supplied by Native Plant lnc.( Salt Lake City, Utah, USA). The inoculum was blended with a small amount of soil to release the spores from roots and to facilitate uniform distribution in the soil mix. Spores of the fungus were then incorporated throughly into the soil mix at rates sufficient to achieve a final spore density of 10 spores g" in all experiments. 97 Final spore densities were determined by wet-sieving (Gerdemann and Nicolson, 1963), sucrose centrifugation and then counting spore propagules under a dissecting microscope. Phenolic compounds The phenolic compounds tested were p-coumaric acid, p-hydroxybenzoic acid and quercetin (Figure 4.1). They were obtained from Sigma Chemical Co., St. Louis, MO, and first dissolved in 1 ml ethanol, then transferred to 250 ml of distilled water. Solvent controls were prepared using similar volumes of ethanol and water. To treat plants, concentrations of 0.25 mM and 1.0 mM were used for each phenolic compound. Solutions were freshly prepared for each application. In all experiments, 10 ml of each test solution was placed in the center of each pct (80 g of sand-soil mix) with a pipette dispenser prior to transplanting. Experiments Several growth chamber experiments were conducted, varying the frequency of application of the phenolic compounds studied. Plants from experiments were harvested three weeks after-emergence. In the first experiment (A), white clover plants received 10 ml of each solution only at the onset of the experiment. In the second experiment (3), 10 ml of each compound was added to the soil every 7 days. In a third experiment (C). the rate of application was for every 4 days. In the fourth experiment (D). Figure 4.1. Structures of pcoumaric acid (3-(4-hydroxyphenyl)-2-propenic acid), p- hydroxybenzoic acid (4-hydroxy) and quercetin (3, 3', 4’, 5, 7 - pentahydroxyflavone). HO O CH=CHCOOH p-Coumaric Acid COOH HO p-Hydroxybenzoic Acid Ouercetln IN the effects of the phenolic compounds on root colonization and growth were examined in sorghum plants. In this experiment, sorghum plants received the initial 10 ml of each solution and then an additional 10 ml was applied every 7 days. In a fifth experiment (E), sorghum plants were grown as in experiment D with the exception that applications of the compounds were at 4 days intervals. Growth conditions After transplanting, plants were kept in a growth chamber with 300 pmol ms of photosynthetic active light with a 14 h day length, 28°C and 21°C day and night temperatures, respectively. Plants were watered once a day with distilled water, except when nutrient solution or phenolic solutions were applied. Nutrient solutions, half-strength Hoagland’s solution (Hoagland and Amon, 1950), were applied as 10 ml per pot, three times a week. At 3 weeks after- emergence, plants were harvested and assessed for growth and root colonization. Shoots were separated from roots, weighed and dried at 70° C for 2 days. Roots were washed free of soil, weighed and dried at 70° C for 2 days. A weighed subsample was removed, cleared and stained with trypan blue (Phillips and Hayman, 1970). Root colonization rates, expressed as percent of root segments colonized by AM fungi, were estimated by the line intersect method (Korrnanik and McGraw, 1982). Experimental design and statistical analysis All experiments were conducted using a completely randomized design 101 with at least 16 plants per treatment, and the experiments were repeated, at least once, with similar results. Data reported here are means of one experiment derived from a complete analysis of variance (ANOVA) using MSTAT statistical package program. Shoot dry weight, root dry weight and root colonization by G. intraradices of white clover and sorghum plants were analysed and compared by Least Significant Difference (LSD) tests (p< 0.05). Each experiment is presented in a separate graph. RESULTS The effects of p-coumaric acid, p-hydroxybenzoic acid and quercetin on growth and root colonization of mycorrhizal white clover plants were assessed when 0.25 mM and 1.0 mM solutions were applied at the beginning of the experiments. At the lower concentration, shoot and root dry weights of phenolic- treated mycorrhizal plants were significantly greater than that of control plants grown in the absence of the compounds (Figure 4.2). Interestingly, quercetin also increased the growth of clover plants (shoot and root) when the soil was amended with the 1.0 mM solution. At this concentration, plants receiving p-coumaric acid and p-hydroxybenzoic acid did not differ from control plants in shoot dry weight, while root growth was greater only in the presence of p-coumaric acid. The Root/Shoot (R/S) ratio of phenolic-treated plants was significantly greater than for control plants at 0.25 mM, while only p-hydroxybenzoic acid was similar to controls when applied at 1.0 mM. 102 Figure 4.2. Growth and root colonization of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied once, at planting, at 0.25 and 1.0 mM solution concentrations (experiment A). Data represent means of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05). I585 mll lby SHOOT onv WEIGHT (mg) noor onv WEIGHT (mg) COLONIZATION (96) N U" N O .5 (D .e O 0‘ 10 80 40 20 0.0 [:1 comm. p-COUMARIC ACID 0.25 103 1 .0 0.25 1 .0 CONCENTRATIONS (mM) 0.25 w p-HYDROXYBENZOIC ACID W/j OUERCETIN 1.0 The l slgnl lilgt color sllml soll." Dian the eel: 30? WE Si; 104 The percent of root colonization in plants that received phenolic compounds was significantly greater than mycorrhizal control roots at both concentrations used (Figure 4.2). P-hydroxybenzoic acid and quercetin stimulated the root colonization at the lower concentration (53 and 57 %, respectively). The stimulation by these two compounds was slightly decreased when the 1.0 mM solution was applied (43 °/o and 44 °/o, respectively) and only quercetin-treated plants were able to improve the growth of white clover. P-coumaric acid showed the lowest root colonization when it was applied at 1.0 mM (25%). When white clover was treated with p-coumaric acid, p-hydroxybenzoic acid or quercetin every 7 days, shoot dry weight was not affected by the application of the phenolics at 0.25 mM (Figure 4.3). When these compounds were applied at 1.0 mM, the growth of the plants was significantly less than that of the controls (30 % less) and also less than phenolic-treated plants at 0.25 mM. Plants treated with p-hydroxybenzoic acid at 0.25 mM had the greatest root growth, but growth greatly decreased when applied at 1.0 mM, followed by quercetin. No significant differences in root to shoot (R/S) ratios were found in this experiment. The percent root colonization was higher when p-coumaric acid and p- hydroxybenzoic acid were applied at 0.25 mM (Figure 4.3). At 1.0 mM, these compounds were less stimulatory. The plants given quercetin at 0.25 mM had root colonization level similar to those of control plants, whereas at 1.0 mM it significantly inhibited the percent of root colonized by G. intraradices (18 %). 105 Figure 4.3. Growth and root colonization of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied 3 times, 7 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment 8). Data represent means of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05). YDR XYBENZOIC ACID 0 H R eeeeeeeeeeeeeeeeeeeee ooooooooooooooooooooo ooooooooooooooooooooo OOOOOOOOOOOOOOOOOOOOOO 000000000000000000000 000000000000000000000 ooooooooooooooooooooo // ERETI N C U 0 ooooooooooooooooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooooooooooooooooo oooooooooo ooooooooooooooooooo ............ 30.3.40...o...o.o..o.0.e.e.e.coca... . .... OOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO oooooooooooooooooooooooooooooo oooooooooooooooooooooooooooo e%//////////H I. 0000000000 oooooooooooooooo 00000000000000000 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOO 0000000000000000 OOOOOOOOOOOOOOOOO 000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000 ...... . we”. ”.uonouonou. ” on .Hc”guano“.uonoHe”one"anew-mameuoueuaeooeuououo”an.“ oooooooooooooooooooooooooooooooooooooo ooooooooooooooooooooooooooooooo 000000000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000000000000000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000000000000 .................................. .............................. oooooooooooooooooooooooooooooo eeeeeeeeeeeeeeeeeeeeeeeeeeeeee oooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooo OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO oooooooooooooooooooooooooooooooooooo oooooooooooooooooooooooooooooooooooo OOOOOOOOOOOOOOOOOOOOOOOOOOO 0000000000000000000000000000 OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 000000000000000000000000000 ....................................................... 1 OOOOOOOOOOOOOOOOO 000000000000000000000000000000000000000 00000000000000000 .......... O .0. 000.000.0000.". H” OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO _ m days lllClEE andi mycc acid weir deer 107 In a subsequent experiment, phenolic compounds were applied every 4 days (Figure 4.4). Only p-hydroxybenzoic-treated plants showed a significant increase in shoot dry weight at 0.25 mM (44% over the control). P-coumaric acid and quercetin had no effect on plant dry weight, at this concentration. At 1.0 mM, all compounds significantly decreased shoot growth compared to mycorrhizal controls. Root growth was significantly affected by application of p-hydroxybenzoic acid (Figure 4.4). At 0.25 mM, p-hydroxybenzoic had the highest root dry weight. At 1.0 mM, p-coumaric acid, had the lowest root dry weight (39 % decrease). At this concentration, p-hydroxybenzoic acid and quercetin-treated plants resulted in the greatest R/S ratios, which could be due to the decreases in shoot growth while root growth rates were similar to the controls. P-coumaric and p-hydroxybenzoic acids improved root colonization (approximately 45 % each) at 0.25 mM (Figure 4.4). At this concentration, colonization levels of quercetin-treated plants did not differ from control roots. When applied at 1.0 mM, all the exogenously-applied compounds significantly reduced root colonization. P-hydroxybenzoic and quercetin-treated plants caused a 43 % decrease in root colonization compared to the control, and p- coumaric acid caused a decrease of 26%. Phenolic compounds were exogenously applied to sorghum plants every 7 days (Figure 4.5). All compounds at 0.25 mM improved the growth of the D|ants in both shoot and root dry weight. P-coumaric acid significantly increased shoot dry weight (19 %) followed by p-hydroxybenzoic acid (14 %) and to a 108 Figure 4.4. Growth and root colonization of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied 5 times, 4 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment C). Data represent means of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05). SHOOT onv WEIGHT (mg) noor onv WEIGHT (mg) COLONIZATION (x) D CONTROL N p-COUMARIC ACID 7% OUERCETIN 0.0 0.25 109 m p-HYDROXYBENZOIC ACID 1 .0 0.25 1 .0 0.25 1 .0 CONCENTRATIONS (mM) 110 Figure 4.5. Growth and root colonization of inoculated (Glomus intraradices) sorghum plants as affected by different phenolic compounds applied 3 times. 7 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment D). Data represent means of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD(p<0.05). .W//////////////%m .................... .................... ...................... ......................... // /////////////// //////////////// / nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn / ................................... ............... %U V 2.3.“.n.................u.n.3:.u.u...a".u.u.u.u...n.....u...u. .................................... ........................ . ......................................... .///////// ./////////////// .................................................. ................................................. ....... ........................................... a; .............. ............ .......................................... ......... ...... .......... ................ ...... .......... ....... ............ ................ ................................................. ................................................................. ...................... ...n.u...u.u.. .6..o......o.......u.n.u... n.u.....u............o ..... .................................... .............................. ............... ........................................................... ............................................................. ........................................................................................................................... ....................................... ...................................................................... “Amman.” ............................................................................ ............................................................................ ...................................... \\ 112 lesser extent quercetin (7 %) treated plants, which did not differ from control plants. Root dry weights were significantly increased by p-hydroxybenzoic acid (42 %), p-coumaric acid (19 %) and quercetin (11 %). The increase in root growth by p-hydroxybenzoic acid, at 0.25 mM, led to an increased R/S ratio. At 1.0 mM, the treatments of phenolic compounds did not have any effect on shoot and root dry weights. All compounds significantly stimulated root colonization by Glomus intraradices, at 0.25 mM (Figure 4.5). Stimulation of relative root colonizations (72%) by 0.25 mM p-coumaric acid led to a significant increase in plant growth, while at 1.0 mM, p-coumaric acid had no effect. Only p-hydroxybenzoic acid at 1.0 mM increased root colonization, but sorghum growth was unaffected. When the phenolic compounds were applied every 4 days post- emergence (Figure 4.6), the shoot dry weights of treated plants were not stimulated by any of the compounds. At the lowest concentration, shoot dry weights were similar to those of controls for p-coumaric acid-treated plants, and lower when either p-hydroxybenzoic acid or quercetin was amended to the soil. Root growth, at this concentration, was similar to that of control roots for all phenolic-treated plants. At 1.0 mM, all compounds were inhibitory to shoot and root growth. The R/S ratio was not affected by any of the above compounds tested. P-hydroxybenzoic acid significantly increased root colonization by G. intraradices, when applied at the lower concentration (Figure 4.6). P-coumaric acid and quercetin did not affect colonization at the lower concentration. At the 113 Figure 4.6. Growth and root colonization of inoculated (Glomus intraradices) clover plants as affected by different phenolic compounds applied 5 times, 4 days intervals, at 0.25 and 1.0 mM solution concentrations (experiment E). Data represent means of 12 plants with standard errors. Means followed by the same letter within each graph are not significantly different by LSD (p<0.05). SHOOT onv WEIGHT (mg) noor on)! WEIGHT (mg) COLONIZATION (7.) 114 120 [:j CONTROL m p-HYDROXYBENZOIC ACID N pCOUMARIC ACID % OUERCETIN 90 be 30 60 30 30 20 10 0.0 0.25 1.0 0.25 1.0 0.25 1.0 coucsummous (mM) 115 higher concentration (1.0 mM), a significant decrease in root colonization was observed in all phenolic-treated plants. DISCUSSION This is the first report involving p-coumaric acid, p-hydroxybenzoic acid, and quercetin on the establishment of mycorrhizae in white clover and sorghum roots. The establishment of mycorrhizas and plant development depended greatly upon the presence, the number of applications and the solution concentrations of applied phenolic compounds in the soil. In general, the lower solution concentrations (0.25 mM) stimulated plant growth and root colonization, whereas at higher solution concentrations (1.0 mM), inhibitory effects were observed. All compounds stimulated clover root colonization by Glomus intraradices when applied once in the soil, at both low and high concentrations (Figure 4.2). The plant growth response was observed when these compounds were applied only at 0.25 mM, except with quercetin, which also stimulated white clover plant growth at higher concentrations. When the soil applications of these compounds were increased to every 7 or 4 days, only the phenolic acids, p-coumaric and p-hydroxybenzoic, stimulated root colonization when applied at 0.25 mM. The flavone quercetin did not have any effect on root colonization at this concentration. Sorghum plants also were stimulated by all compounds when applied at lower concentrations every 7 days, but only p-coumaric acid and 116 p-hydroxybenzoic acid were able to improve the growth of the plants. Applications every 4 days caused a decrease in root colonization, except with p- hydroxybenzoic acid. The growth of the plants was also affected by the presence of the compounds. There was a tendency for all compounds to be inhibitory to both root colonization and growth of both clover and sorghum at higher concentrations (1.0 mM), when applied every 7 or 4 days. As in other symbiotic systems (Lynn and Chang, 1990), phenolics may play a role in signalling the events that lead to the various physiological and anatomical changes in an AM symbiosis. The involvement of plant signals and recognition mechanism in arbuscular mycorriza is supported by the findings that the presence of roots (Mosse, 1988; Becard and Piche, 1989, 1990), root exudates (Elias and Safir, 1987; Graham, 1982), legume seed diffusates (Hepper, 1979), suspension cultured legume cell and cell products (Carr et al., 1985; Paula and Siqueira, 1990) all stimulated the hyphal growth of free-living mycelium. Recently, it has been shown that isoflavonoids, such as fcrrnononetin and biochanin A, isolated and identified from stressed clover roots (Nair et al., 1991) can be inferred as AM-stimulating compounds, at low concentrations (Siqueira et al., 1991b). Another class of phenolics has been shown to influence mycorrhizal spore germination and hyphal growth in vitro. Tsai and Phillips (1991) added from 0.0 to 10 pM of quercetin to an in vitro assay and found that this compound, only at 2.5 pM, promoted three developmental processes in AM fungi G. etunicatum: spore germination, hyphal elongation, and hyphal branching. Quercetin was added only at the beginning of the experiment, rather 117 than after germination, and so it was suggested that the very rapid quercetin- induced increase in spore germination contributed to the promotion of hyphal growth and branching. Becard et al. (1992), tested various flavonoids for their ability to stimulate in vitro growth of germinated spores of AM fungi. Generally, they found inhibition using these compounds, but quercetin (10 pM) prolonged hyphal growth from germinated spores of Gigaspora margarita and G. etunicatum. Therefore, quercetin may be able to influence this first stage of mycorrhizal formation. In both studies, quercetin exhibited a pronounced concentration effect with regard to its impact on these developmental processes (T sat and Phillips, 1991; Becard et al., 1992). In our study, quercetin was able to improve root colonization of white clover plants, only when applied at the beginning of the experiment (Figure 4.2) at both concentrations used. Increasing the number of soil applications, reduced or eliminated this effect of quercetin. Thus, as with the above in vitro assays. quercetin seems to have a very narrow range of concentrations, which enhance either the stimulation of spore germination, hyphal growth and branching or the effectiveness of the Trifolium-Glomus symbiosis, irrespective of the type of fungi present. Therefore, quercetin could be an important molecule involved in the host-recognition mechanism in AM symbiosis, especially in legume plants. When applied in another system (Sorghum-Glomus), root colonization stimulation by quercetin at 0.25 mM was smaller than that of the phenolic acids, at 0.25 mM, and did not improve the growth of sorghum plants. Furthermore, stimulation of growth of the VAM fungi by quercetin was observed, but the 118 effectiveness of the symbiosis was impaired when compared to that of the phenolic acids. Since flavonoids are found in low concentrations in soils, primarily in legume planted soils, their effect on VAM symbiosis of gramineous plants may not be as relevant as it is on leguminous plants. Becard and Piche (1989) have developed a model and proposed two different mechanisms for the formation of the VAM symbioses. Each mechanism is triggered by root factors during the growth of the fungi. The first mechanism (M1) is responsible for the stimulation of hyphal growth from a germinating spore where the fungus is still dependent on spore reserves for growth and development. They proposed that certain root exudates regulate the ability of the fungus to use its endogenous reserves. The second mechanism (M2) is triggered during the formation of arbuscules. At this stage, the hyphal growth becomes dependent on the host and no longer requires the presence of the spores. Therefore, quercetin, in the presence of gramineous plants, may stimulate the M1 mechanism, but M2 may be regulated by the host plant or be sensitive to the concentration of this compound. There are different stages throughout the process of the interaction between roots and mycorrhizal fungi during which the process may be halted. Under the conditions of our experiments, quercetin was not tested on sorghum plants when applied only once, at the beginning. If quercetin is involved in the stimulatory effects of AM fungi, like the isoflavone formononetin, there is a possibility that in small concentrations, stimulatory effects on growth and root colonization of sorghum plants could occur. The mechanisms by which quercetin stimulates spore germination, hyphal growth and branching (T sai et 119 al., 1991; Becard et al.,1992) and root colonization (as reported here) cannot be determined from these experiments. Phenolic acids, another group of biologically active phenolics, have been isolated from plants and soils. Caffeic, ferulic, p-coumaric, p-hydroxybenzoic and vanillic acids appear to be the most persistent (Whitehead, 1964; Lodhi, 1975). Ferulic and p-coumaric acids were commonly found in residues of corn, oats, sorghum and wheat as well as in the soils in which these crops were grown (Guenzi and McCalla, 1966). These compounds have been studied because of their association with allelopathic interactions in some ecosystems and in cultivated soils (Wang et al., 1967; Patrick, 1971; Blum and Shafer, 1988). Among the phenolic acids, caffeic and ferulic acids have also been reported to influence the AM symbiosis between Glomus fasciculatum and asparagus plants. Pederson et al. (1991) observed a linear increase of inhibitory effects on AM root colonization with increasing concentrations of these phenolic acids. Additionally, extracts from asparagus soils were shown to be inhibitory to the development of the mycorrhiza, but not in those treated with the non-asparagus soil extract or in the control. Pederson et al. (1991) conclude that these phenolic acids, produced by asparagus plants, could be important components in the asparagus decline problem. In our study, p-coumaric acid, showed a stimulatory effect on growth and root colonization of sorghum plants, only when applied weekly, at 0.25 mM (Figure 4.5). At higher concentrations, only when applied every 4 days, a pronounced inhibitory effect was observed (Figure 4.6). It has been reported 120 that sorghum plants exude large amounts of p-coumaric acid into the soil. Guenzi and McCalla (1966) estimated that the residue from a single crop of sorghum could return about 100 Kg.ha" of p-coumaric acid to the soil. Therefore, the effect of p-coumaric acid on growth and colonization of sorghum plants was expected to be inhibitory only at the highest concentrations in the soil. Nutrient applications have been shown to reduce the phytotoxic effects of phenolics (Stowe and Osborn, 1980; Blum and Shafer, 1988; Pederson at al., 1991). This may partially explain the behavior of p-coumaric acid observed in our study, in that nutrient solutions were applied three times a week. Another possibility could be that phytotoxic levels of these compounds may be alleviated by the presence of AM fungi, as suggested in other studies (Leake et al., 1989: Pederson etal., 1991; Siqueira etal., 1991c). Interestingly, when p-coumaric acid was applied at the lower concentration to clover plants. stimulation of root colonization was observed in both 7 and 4 days applications (Figures 4.5 and 4.6). but did not affect the growth of the plants. Although the higher concentration, applied weekly, did not affect root colonization, growth inhibition was observed when applied at 4 days intervals, where inhibition on root colonization also occurred. A similar effect was observed with p-hydroxybenzoic acid. Since these compounds are commonly found in soils, and arbuscular mycorrhizal (AM) symbiosis is ubiquitous in the plant kingdom, it is likely that lower concentrations can stimulate the growth of AM fungi, but the response on growth will depend on the AM host. Although AM fungi are typically considered to have low or no host 121 specificity, there is increasing evidence that some degree of specificity exists between AM fungi and plants (Krishna et al., 1985; Azcon and Ocampo. 1981; Estaun et al., 1987; Rosendahl et al., 1990). Host genotype was a major factor in the establishment of AM colonization and its effectiveness. Krishna et al. (1985) detected significant differences in colonization potential of 30 genotypes of pearl millet, which ranged from 25 to 56%, and they suggested that the formation of phenolics and phytoalexins could be involved. Azcon and Ocampo (1981) correlated differences in AM infection of 13 wheat cultivars with the level of soluble sugars in root exudates. In pea cultivars, three Glomus species infected similar root lenghts, but the degree of stimulated root growth and interaction with phosphate were not correlated (Estaun et al., 1987). Therefore, even within the same plant genera, differences in colonization and the effectiveness of the symbiosis have been seen. Presently, it is very difficult to link a specific responses of AM mycorrhiza to a specific chemical, but considering the coevolution of plants and AM fungi, it may be possible that some phenolic compounds exuded or synthesized by specific plants have a powerful influence on the outcome of the symbioses. Giovannetti et al. (1994) outlined at least three sequential steps in the recognition process of AM symbioses. Their study used an VAM host, a non- VAM host and a non-mycorrhizal host. They observed that only the perception of the right chemical signals, coming from the roots of host plants, promote a differential morphogenesis of VAM hyphal growth (branching and proliferation in all directions), presumably to increase the chance of the hyphae encoutering a 122 root. This was not observed in non-mycorrhizal or non-arbuscular mycorrhiza hosts. After appressorium formation, root cell penetration by the fungus depended on the genome of host plants.Therefore, the effects of phenolic acids applied to clover plants, could trigger mycorrhizal formation, but the effectiveness of the VAM colonization is later impaired. Also these compounds, in the presence of a legume plant, apparently stimulated the M1 mechanism, proposed by Becard and Piche (1989), but the second mechanism, was likely to be regulated by the host plant in response to the presence or uptake of these compounds. Inhibition of growth and root colonization were observed when phenolics were applied to the soil at higher concentrations. In field situations, phenolics can build up in soils and selectively influence soil microbial populations (Hartley and Whitehead, 1985). Studies have shown the effects of plant-produced allelochemicals on AM fungi (Wacker et al., 1990a). Shifts of VAM fungal populations can negatively affect the growth of crop plants in the field, and may be related to the accumulation of the phenolic compounds (Wacker et al., 1990a) Monocropping systems of asparagus plants have been associated with decrease in root colonization with a concomitant decrease in plant growth (Walker et al. 1990a, 1990b; Pederson et al., 1991). Recently, Vivekanandan and Fixen (1991) observed that similar levels of root colonization occurred in the com-soybean rotation and corn monocrop systems, but significant differences occurredin plant growth and P uptake between these systems. When com- 123 soybean rotations were applied, increased growth and P uptake of the plants was observed. Corn monocropping significantly decreased these parameters. Additionally, Johnson et al. (1992) reported that continuous monocultures of both corn and soybean generally had lower yields and tissue concentrations of P, Cu, and Zn than when com or soybean were grown in rotation systems. They suggested a mechanism to explain how AM fungi could cause yield depressions associated with monoculture. Again, a shift in VAM populations has been proposed, in that selection against the most rapidly growing and sporulating fungal species occurs and the detrimental species (inferior mutualists or even parasitics) increase. Interruption of monoculture would increase the relative abundance of beneficial fungi over the detrimental ones. Monocropping systems also allow the build up phenolic compounds in the soil (Siqueira et al., 1991a), which in turn also may have an impact on soil microorganisms, including VAM fungi In summary, our study suggests that phenolic acids can affect root colonization as well as plant growth and that specific plant metabolites could be active in the soil. The build up of these compounds in the soil could have negative effects on mycorrhizal symbiosis. Phenolic acids, commonly found in soils, seems to have less impact on VAM symbiosis, whereas quercetin, seems to have a more specific role in the regulation of VAM symbiosis. Plants and their mycorrhizal fungi have coevolved to form a valuable relationship. Phenollcs are among the most widespread classes of secondary metabolites produced by plants and AM fungi can form symbiotic associations 124 with most crop species. Therefore, the development of the mycorrhiza and the involvement of phenolics as regulatory molecules should not be overlooked. Studies on VAM symbiosis under monocropping or rotation systems could include a quantification of the active phenolics present in the soils which could improve our potential to use these beneficial fungi more effectively in developing agricultural systems. LIST OF REFERENCES Azcon, R. and Ocampo, J. A. 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R. and Stephenson S. N. (1990b) Evidence for succession of mycorrhizal fungi in Michigan asparagus fields. Acta Horticulture 271, 273- 279. Wang, T. S. C., Yang, T. K and Chuang, T. T. (1967) Soil phenolic acids as plant growth inhibitors. Soil Science 103, 239- 245. 130 Whitehead, D. C. (1964) Identification of p-hydroxybenzoic, vanillic, p-coumaric and ferulic acids in soils. Soil Science 202, 417- 418. CHAPTER 5 SUMMARY AND CONCLUSIONS Activity and isozyme patterns of NAD-malate dehydrogenase, esterase. and peroxidase were investigated in a Zea mays-Glomus intraradices symbiosis at early stages of development, in the presence or absence of formononetin, a mycorrhiza-stimulatory isoflavone. At two weeks of growth, formononetin-treated mycorrhizal roots had a sharply increased fungal colonization (80%), whereas the effect diminuished as the symbiosis matured (15%). Forrnononetin, an isoflavone, was isolated and identified from stressed clover plants (Nair et al., 1991). This compound increased both hyphal growth in vitro and root colonization by G. intraradices in clover (Siqueira et al., 1991). The mechanisms for stimulating fungal growth and colonization are yet to be determined. Mycorrhizal roots increased expression of two NAD-malate dehydrogenase (MDH) isozymes at three weeks of growth. The exogenously applied formononetin further enhanced the activity of these isozymes. MDH4 was strongly expressed only in VAM roots, which suggests that MDH activity is probably linked to the amount of fungal biomass in VAM roots, since no differences were observed in root growth and protein content between VAM and non-VAM roots. In addition, the activity of one MDH isozyme was observed only 131 s.» . 132 in VAM roots, growing under different phosphorus (P) levels in the soil. MDH isozymes have been used previously for the identification of VAM fungi in mature symbiosis of leek roots (Rosendahl, 1992). Fungal biomass was not separated from the root tissue in our study, but the results suggested that MDH isozyme activities observed were in response to active mycorrhizal colonization. In this regard, the isozymes MDH4 and MDH3 observed in our experiments, only in VAM roots, could possibly be used as markers of fungal biomass in the roots at early stages of mycorrhizal symbiosis. Esterase (EST) isozyme activities did not follow the same time frame as did malate dehydrogenase. At two weeks of growth, the activities of several esterase isozymes were enhanced in mycorrhizal roots. At this time, root colonization increased dramatically (80%) in roots treated with formononetin and this increase was reflected in an increase in the expression of these isozymes. At three weeks, no differences in EST activity were detected between VAM and non-VAM roots. When esterase isozymes were studied under different levels of P in the soil, at three weeks of growth, no differences were observed between VAM and non-VAM roots, despite the increased level of available P in the soil. Esterase isozymes have been shown to be more important to VAM symbiosis at very early stages of the symbiosis, and unlike MDH, EST is correlated with rapid increases in root colonization. Further characterization of this EST enzyme system could be useful in studies on the competition between different VAM fungi for specific host roots. 133 The host peroxidase reaction to the presence of VAM fungi was observed at early stages of the mycrosymbiont-host recognition. At one week of growth. VAM roots had increased peroxidase (POX) activity, which dramatically decreased in the presence of formononetin at two weeks of growth. At this time, two weeks, mycorrhizal plants maintained a higher level of POX activity compared to non-VAM plants. The increase in root colonization was also dramatic for fcrrnononetin-treated plants at two weeks of growth, suggesting that the presence of formononetin supressed the activity of POX, facilitating the penetration and fungal spread. At three weeks, total POX activity in VAM roots decreased below the levels of non-VAM roots, while formononetin-treated VAM roots further decreased. Moreover, when com plants were grown under different levels of available soil P, peroxidase activity increased with increasing levels of soil P in the soil. In low to moderate soil P VAM roots had lower POX activity than non-VAM roots, which correlates with the high root colonization at these P levels. The presence of formononetin in VAM roots further decreased the activity of this enzyme compared to VAM alone. Interestingly, formononetin- treated non-VAM roots also had decreased POX activity. The results suggest a possible mechanism of action for the formononetin-stimulatory effects on fungal colonization. Since formononetin alone also decreased POX activity at all soil P levels, VAM fungi may take the advantage of the lower peroxidase activity expressed in these roots to rapidly penetrate the root cortex. Activity and isozyme patterns of acid phosphatase (ACP), and alkaline phosphatase (ALP), were also investigated under different soil phosphorus (P) 134 levels in a three-week-old Zea mays-Glomus intraradices symbiosis. Alterations in ACP and ALP activity were dependent on the level of available soil P. VAM roots had higher ACP and ALP activities at the low levels of soil P availability. Forrnononetin-treated VAM plants further increased these activities. Additionally, the response of these enzymes was closely related to root colonization. The increase in ACP and ALP enzymes associated with VAM fungal colonization may be related to the role of mycorrhizal colonization in improving P concentration of plants colonized. ACP activity was more responsive to the different levels of P in the soil, which suggests an involvement in “mining” the P in the soil, while ALP could be involved in the active phosphate assimilation in mycorrhizal roots. Therefore, monitoring specific enzyme systems during the establishment of VAM symbiosis has given us useful tools for studies with endomycorrhizae. Also, it is recomended that physiological studies involving mycorrhizal associations should always consider the level of available soil P, because it is difficult to compare results when the soil P levels are not considered or are different. The effects of p-coumaric acid, p-hydroxybenzoic acid, and quercetin application on growth and colonization of clover (Trifolium repens L., cv Ladino) and sorghum (Sorghum bicolor L.) roots by the VAM fungus Glomus intraradices were determined, using 0.25 and 1.0 mM solutions of these phenolics. The rate of application of these compounds was also varied. In general, lower concentrations (0.25 mM) of the phenolic compounds were able to stimulate 135 plant growth and fungal colonization, whereas at higher concentrations (1.0 mM), inhibition occurred. The accumulation of these compounds in the soil negatively affected the establishment of VAM symbioses in both clover and sorghum roots. The results suggest that phenolic acids have limited influence on the establishment of mycorrhizal symbioses, whereas quercetin is likely to have a larger role in the regulation of VAM associations, especially in leguminous plants. The results are discussed in relation to the recognition process, host specificity, and the cropping systems involved. Studies involving phenolic compounds in relation to mycorrhizal associations may provide new insights concerning the behavior of these obligate symbionts. These studies may also lead to the potential use of phenolic compounds as soil amendments to exploit the indigenous populations of VAM fungi. 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