Effect of Vesicular-Arbuscular Mycorrhiza on phosphorus metabolism in agricultural plants

Microbiol. Res. (1994) 149, l39-143

Microbiological Research ©

Gustav Fischer Verlag Jena

Effect of Vesicular-Arbuscular Mycorrhiza on phosphorus metabolism in agricultural plants A. V. Shnyreva 1, I. S. Kulaev 2 1 2

Department of Mycology and Algology, Faculty of Biology, Moscow State University, 119899 Moscow, Russia Department of Molecular Biology, Faculty of Biology, Moscow State University, 119899 Moscow, Russia

Accepted: December 15, 1993

Abstract The effect of VA-mycorrhization of corn plants by fungi of Glomus spp. on phosphorus metabolism has been studied. Phosphorus content in the VA-mycorrhizal corn root tissues increased by 35% for the species Glomus mosseae and by 98% for Glomus fasciculatum. Phosphorus was accumulated in the root tissues as low-molecular organophosphorus compounds of the acid-soluble fraction and high-molecular compounds of the acid-insoluble fraction (RNAs). Daily dynamics of phosphorus transfer to the above ground parts of the mycorrhizal corn plants depended on the stage of plant vegetation: by the 60th day phosphorus carry-over was predominantly as orthophosphates, whereas the 70-day-old corn plants showed a significant increase of the organophosphorus content in the daily root exudate. Phosphorus nutrition of corn plants has been shown to be an essential factor of biosynthetic processes in the plant. Root infection by VAM fungi has greatly improved the phosphorus uptake, translocation and its subsequent transfer through the host plant.

Key words: VA-mycorrhiza - phosphorus accumulation and transfer

Introduction Mycorrhiza, the symbiotic associations between soil fungi and host plant roots are widely spread both in nature and agrocenoses. It is well known that vesicular-arbuscular mycorrhizal (V AM) fungi benefit the host plant primarily by increasing the capability of the root system to absorb and translocate Corresponding author: I. S. Kulaev

phosphorus (P) and minor elements through an extensive network of hyphae external to the root (Bolan et al. 1984; Fitter 1991). The enhanced growth rates of plants infected by VA mycorrhizal fungi are currently believed to result from improved nutritional status of the host. On the other hand, the high level of soil P somewhat limits VAM infection. According to some authors (Graham et al. 1981; Garriok et al. 1989), P concentration in plant tissues is one of the factors controlling mycorrhizal symbiosis, and the regulation mechanism is associated with permeability changes of root tissue membranes. The biochemical mechanisms of active phosphorus transport by the VAM fungal component and subsequent phosphorus translocation into host cells have been studied inadequately. Most orthophosphates absorbed by infected roots have been shown to be converted into condensed polyphosphates (Cox et al. 1980); the polyphosphates are synthesized in the VAM fungus and, being transported by the cytoplasmic streaming to the arbuscules, are converted by either an enzyme of polyphosphatase type, releasing orthophosphates, or a polyphosphate kinase releasing ATP. The products released are then either directly or indirectly used in translocation of the orthophosphates across the host-arbuscule interface. An alternative way is that the polyphosphates can be involved in phosphorylation mechanisms required for active carbohydrate transport from the host roots into the arbuscules (Newman and Ritz 1986; Smith et al. 1991). The object of this paper is, first, to demonstrate the effect of mycorrhization on phosphorus metabolism in agricultural plants, particularly corn, and, second, to obtain more complete data on biochemical Microbiol. Res. 149 (1994) 2

139

processes occuring in infected host tissues under increased phosphorus accumulation and subsequent transfer through the plant.

Material and methods Source of VA mycorrhizal cultures. We studied two species of Endogonaceae mycorrhizal fungi: Glomus mosseae (Nic. et Gerd.) Gerd et Trappe and Glomus fasciculatum (Thaxter sensu Gerd./Gerd. et Trappe) isolated from mycorrhized corns roots. Both VAM cultures were obtained from Research Institute of Agricultural Microbiology, St. Petersburg. Growth and mycorrhizing plants. Seeds of corn (Zea mays L. cv. 'Collectivny') were surface-sterilized and germinated on water agar (1.5%). The experimental soil was prepared by mixing two parts of soil with one part of sand (w/w) and sterilized (at 101°,4 h) to kill the mycorrhizal fungi indigenous to the soil. The soil was fertilized with insoluble phosphorus - oxyapatite (at the rate of 0.5 g per 7 kg size pot). Inoculation mixture (1.5 g of comminuted mycorrhized corn roots mixed with 100 g of sterilized sand) was introduced into pots to the depth of 3 cm below the substrate surface. Germinated seeds were transfered into moist soil and covered with a thin layer of sand. Pot cultures (corn seedlings) were grown under 16 h light, and 26 - 28° /15 °C day and night temperatures respectively. Plant were watered daily to maintain 60% humidity and fed weekly with 400 ml/pot of a Mosse mineral mixture (Mosse 1962) and at 20 and 40 d with 15 ml/pot of a Klein element mixture (Menge and Timmer 1982). There were 3 sets of experiments, one with corn plants mycorrhized by Glomus mosseae, the other with corn plants mycorrhized by Glomusfasciculatum culture and the third with non-mycorrhized host plants (control plants). Each combination was taken in 10 repeats. Sampling and analyses. The plants were collected at 60 and 70 days after emergence of seedlings. Plant dry weights were registered simultaneously at 70 d of vegetation. Mycorrhizal infection was estimated microscopically after staining substrate-washed roots with Aniline blue (Phillips and Hayman 1970) using a lower concentration of KOH (5%) to prevent root disintegration. Plant tissue phosphorus determination and fractionation of phosphorus. Phosphorus (P) compounds from root tissues were isolated and fractionated according to the Langen-Liss method modified by Kulaev (Small Practical Course on Biochemistry 1979) (Fig. 1). At the first stage acid-soluble P fraction (Past) was extracted with cool 0.5 N perchloric acid

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Microbio!. Res. 149 (1994) 2

and then acid-insoluble P compounds (Pait) were fractionated. Phospholipids of the Paif fraction were removed by ethanol-ether extraction. Subsequent fractionation of he acid-insoluble P fraction by the Schmidt-Tanhauser method consisted in separating the DNA and RNA fractions by their different resistance to alkaline hydrolysis. All the P fractions were quantitatively assessed for their Pi content using the Behrenblum-Chain method. Pi was determined by the reduction of phosphomolybdic acid using tin chloride. Exudate collection. At 60 and 70 d of vegetation exudates were collected from the root of corn plants to assess the dynamics of phosphorus transfer to the aerial part of the plant. The plants were cut 24 h after the dressing (at 5 p.m.); the root exudates were collected at 9 p.m. of the same day and at 8 a.m., mid-day and 5 p.m. of the following day, i.e. each 4 hours at the day time and summarily for the night. The exudate volume was immediately measured and Pi content in aliquouts was determined as described above. The remaining portion of the root exudate was used to determine the total P content (Pt) by incineration in 57% perchloric acid at 130°C. The measurements were carried out in triplicates. Separation of RNAs. Total RNA was isolated from corn root tissues by a hot phenol procedure. RNAs were separated by electophoresis on a 12% (w/v) acrylamide gel using 20 x 20 x 0.3 em/vertical plates in Tris-glycine buffer, pH 8.4 (Rubin 1975). The gels were stained with 0.2% aqueous solution of Methylene Blue. Before being loaded on the gel, RNA preparations were denatured by 5 M urea. Statistical analysis. The data from each experiment were analysed employing ST ATGRAPHICS comorthophosphate (Pi)

r

I::l

"d-soluble P (Pasf) 0 5 N perchloric acid

/

acid-insoluble (Paif)

I

ethanol-ether Aaction SOIUblr fraction

insollble fraction alkaline hydrolysis

lipid P RNA-P

Fig. 1. Diagramm of the phosphorus fractionation.

DNA-P

puter package. Statistical analysis by standard error of the mean or significant difference .was conducted where appropriate.

Results and discussion In the present study, considerable VAM infection occured in the corn plants mycorrhized by Glomus fasciculatum (80 - 90%) which was more than three times that under VA mycorrhizal infection by Glomus mosseae (Table 1). No root infection was observed in the plants grown under non-mycorrhizal conditions (uninoculated control samples). This confirmed the sterility of pot cultures in the experiment. Marked differences were observed between the shoot dry weights of plants mycorrhized by G.fasciculatum and that of G. mosseae-mycorrhizal plants at the florification phase (at 70 d): the green biomass increment was 56% more compared with non-mycorrhizal plants) under G.fasciculatum mycorrhization and 10% more under G. mosseae one (Table 1). It should be mentioned that considerable increment of shoot dry matter content of G. fasciculatummycorrhizal plants is due to not only more developed

leaves but more intensive growth of stem and panicle. Dry weight of aerial parts of G. mosseae-mycorrhizal plants was also greater but the difference was not statistically significant. It is thus concluded that the better growth of the mycorrhizal plants was definitely the outcome of the beneficial effect ofVAM association. The experiment was conducted twice with similar results (the previous experiment was carried out by Gorskova and Kulaev (1986)). Both species of the endogonous fungi promoted intensification of organogenesis of the host plant and P accumulation in its root tissues. As seen from Table 2, P content in the corn roots mycorrhized with G. mosseae exceeded that in the control (non-mycorrhizal plants) by 35%, and in the plants mycorrhized with G.fasciculatum it was by 98% higher than in the non-mycorrhizal ones. Thus, root infection by both VAM fungi greatly improved P uptake by corn plants, and P accumulation in the root tissues was acchieved by incorporation into the P-containing compounds. On the whole, the mycorrhizal plants utilize P of fertilizers introduced more efficiently. The analysis of corn root tissues showed accumulation of phosphorus as low-molecular-weight stable

Table 1. Dry matter content in various organs of mycorrhizal corn plants at the panicle earing stage (at 70 d). Dry weight, g' plant -

Mycorrhizal agent

No. of samples

leaves

stems

panicle

total

Root mycorrhization, %

G. mosseae G. Jasciculatum no mycorrhization

9 10 8

6.6a* 9.8b 6.4a

2.2a 2.7b 1.9 a

0.8a l.lb O.4a

9.6a 13.6b 8.7c

20-30 80-90 0

1

* - values represent mean data from five measurements; a, b - means followed by the same letter do not differ significantly (P = 0.05).

Table 2. Content of various P-compounds in corn root tissue. Mycorrhizal agent

Acid-soluble P Pi

Acid-insoluble P Pstab

P content, mgP· g G. mosseae G. Jasciculatum no mycorrhization

0.550 0.540 0.500

1

0.180 0.185 0.060

Pasf

Paif

0.850 0.825 0.620

1.320 0.650 0.480

P total

d. m. * 2.170 1.470 1.100

after collection of the root exudate G. mosseae g. Jasciculatum

no mycorrhization

0.300 0.345 0.315

0.080 0.085 0.065

0.450 0.470 0.440

* root tissue P is expressed as the mean P level of three root systems selected randomly from 10 pots; d. m. = dry matter Microbiol. Res. 149 (1994) 2

141

Table 3. Content of nucleic acids in the root tissues of mycorrhizal and non-mycorrhizal corn plants

Nucleic Content, mg' g-l root d. m. * acid G. mosseae G.fasciculatum no mycorrhization RNA DNA

3.300 0.300

3.700 0.300

2.000 0.300

0.200

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0,150

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0,100

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~ 0,050

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represent mean data from 3 measurements.

organophosphorus compounds of the Pasf fraction (basically, there are free nucleotides) and high-molecular compounds of the Paif fraction (Table 2). The main compounds of the Paiffraction are known to be nucleic acids DNA and RNA. Analysis of the Paif fraction indicated that RNA content in the mycorrhizal plant roots was 1.5 times as high as compared with the non-mycorrhizal ones, whereas DNA content in the root tissues did not vary (Table 3). Thus, it became quite apparent from the present study that activation ofthe host plant root biosynthesis in VAM symbiosis has resulted in intensive use of orthophosphates to construct organophosphorus compounds, primarily RNAs. Similar results have been likewise obtained in the study of ectotrophic mycorrhizae with arboreous species (Harley and McCready 1981). The authors showed the bulk of phosphorus from the culture medium to be transformed into ortophosphates (16.7 -42% of total P) used by the root tissues of the host plants, basically, to form nucleic acids and phospholipids. Analysing rRNA fractions of root tissues, no qualitative differences between VA-mycorrhizal and non-mycorrhizal corn roots could be detected. Therefore , the biosynthetic activity of root tissues has increased under YAM-infection, the effect of the fungi being non-specific. Thus, the analysis of the P-compounds of host root tissues can, in our view, be a qualitative criterion on the assessment of the differences in the biochemical activity of mycorrhizal and non-mycorrhizal com roots as well as other agricultural plants. We likewise studied the dynamics of P transfer to the aboveground part of the host plant. P transfer from the root to the aerial organs was determined as the total P quantity in the entire volume of the root exudate. Daily dynamics of P transport were studied at different stages of vegetation (Fig. 2). At an earlier stage of corn vegetation (at 60 d) the P flow to the aboveground organs of mycorrhizal plants significantly increased during night (1.5 - 2 - fold) and was predominantly represented by orthophosphates (Pi) (Figs. 2 a, 3). At the stem formation stage (at 70 d of vegetation) transfer of P-compounds was the

142

Microbiol. Res. 149 (1994) 2

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0,140 0,120

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non·mycorrflizal

Fig. 2. Daily dynamics of phosphorus transfer into the aboveground part of the corn plants at different seedling ages (all assays were conducted in triplicate and means are shown): A) at 60 d,

B) at 70 d

D Pi .Pt

highest during the day time (Fig. 2 b), the root exudate having predominantly organophosphorus compounds (Po. c.) (Fig. 3). It noteworthy that the daily carry-over of organophosphorus compounds in the 70-day-old mycorrhizal plants was more than four times than that under non-mycorrhizal conditions, while the transfer of orthophosphates (Pi) to the aboveground organs in the mycorrhizal and non-mycorrhizal plants was the same (Fig. 3). Therefore, the increase of P content in the daily root exudates was due to the increment of organophosphorus compounds. It should be noted that in the non-mycorrhizal corn plants the transfer of organophosphorus compounds to the aerial part occured only during the day time. From these results, we have

concluded that increased content of organophosphorus compounds in the root exudates of the VAmycorrhizal plants is correlated with the intensification of biosynthetic processes, including phosphorylation, in the host-plant tissues. These data support our view of VAM fungi activating the biochemical and physiological processes in the host plant root and promoting intensive plant development. In summary, it can be asserted that phosphorus nutrition is an essential factor ofbiosynthetic processes in the plant tissues. VAM fungi considerably activate the root system of the host plant tissues. Root infection by VAM fungi greatly improves the phosphorus uptake from the soil which is due to a more intensive physiological activity of the absorption zone tissues of the corn roots. Phosphorus accumulation in the root tissues of mycorrhizal plants is provided by intensive utilization of orthophosphates to construct high-molecular-weight organophosphorus compounds (mostly RNAs) and is asociated with the change in the transfer of Pcompounds to the aboveground part of the plant. Increased content of RNAs in the corn mycorrhizal root tissues indicates the intensity of the occuring biosynthetic processes. Thus, the present study has allowed to clarify a little the mechanism of P uptake, translocation and transfer in the VA-mycorrhizal plants.

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0,400

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0,200

0

u

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GM

GF

ill K

i

GM

GF

K

70d

60d

days after emergence of seedlings

Fig. 3. Content of P compounds in daily plant root exudate, mg plant (each point is the mean of triplicate determinations): GM - infected by Glomus mosseae, G F - infected by Glomus Jasciculatum, K - non-mycorrhizal roots DPi ~Po.c. .Pt

References Bolan, N. S., Robson, A. D. , Barrow, N . J ., Aylmore, L. A. J. (1984): Specific activity of phosphorus in mycorrhizal and non-mycorrhizal plants in relation to the availability of phosphorus to plants. Soil BioI. Biochem. 16, 299-204. Cox, G ., Moran, K. J. , Sanders, E, Nockolds, C., Tinker, P. B. : Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. 111. Polyphosphate granules and phosphorus Fitter, A. (1991): Costs and benefits of mycorrhizas: implications for functioning under natural conditions. Experientia 47, 350 - 355. Garriok, M., Peterson, R., Ackerley, C. (1989): Early stages in colonization of Allium parrum (leek) roots by the vesicular-arbuscular mycorrhizal fungus Glomus versiforme. New Phytol. 112, 85 - 92. Gorskova, V A., Kulaev, I. S. (1986): VA-mycorrhiza effect on nucleic metabolism in corn roots. Bull. Research Institute of Agricultural Microbiology 44, 10 -14 (in Russ.). Graham, J. H ., Leonhard, R. T., Menge, J. A. (1981): Membrane-mediated decrease in root exudation responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation. Plant Physiol. 68, 548-552. Harley, J. L., McCready, C. C. (1981): Phosphate accumulation in Fagus mycorrhizas. New Phytol. 89, 75 to 80. Menge, J. A., Timmer, L. W. (1982): Procedure for inoculation of plants with vesicular-arbuscular mycorrhizae in the laboratory, greenhouse and field. In : Methods and principles of mycorrhizal research. N. C. Schenk (Ed.). St. Paul, Minn., 47 - 54. Mosseae, B. (1962) : The establishment of vesicular-arbuscular mycorrhiza under aseptic conditions. J. Gen. Microbiol. 27, 509-520. Newman, E. L. , Ritz, K. (1986): Evidence of the pathways of phosphorus transfer between vesicular-arbuscular mycorrhizal plants. New Phytol. 104, 77 - 87. Phillips, J. M ., Hayman, D. C. (1970) : Improved procedure for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. British Mycol. Soc. 55, 158-161. Rubin, G. M . (1975): Preparation of RNA and ribosomes from yeast. In : Methods in cell biology. D. M. Prescott (Ed.), Acad. Press, New York-San Francisco-London, 12, 45 - 64. Small Practical Course on Biochemistry. V Yurkevich (Ed.), Izdatelstvo Moskovskogo Universiteta (1979), (in Russ.). Smith, F., Gianinazzi-Pearson, V, Gianinazzi, S., Smith, S. (1991): Mechanisms of nutrient transfer at the vesiculararbuscular mycorrhizal interface: new insights based on the distribution of A TPases on fungal and plant membranes. In: Root ecology and its practical application. Abstr. ISRR Intern. Soc. of Root Res . 3 Symp., Vienna,

113.

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