Infection by the VA-mycorrhizal fungus

Plant and Soil 103, 213-219 (1987) Martinus NijhoffPublishers, Dordrecht--Printed in the Netherlands

PLSO 7052

Infection by the VA-mycorrhizal fungus Glomus caledonium in Hedysarum coronarium as influenced by host plant and P content of soil LUCIA LIOI l and MANUELA GIOVANNETTI 2 llstituto del Germoplasma, C.N.R., Via Amendola 165/A, 1-70126 Bari, Italy and 2Centro di Studio per la Microbiologia del Suolo, C.N.R., Via del Borghetto 80, 1-56100 Pisa, Italy Received 20 October 1986. RevisedJune 1987

Key words:

Glomus caledonium, Hedysarum coronarium, host-endophyte specificity, VA mycorrhizas

Abstract The low degree of infection of Hedysarum coronarium L. (sulla) exposed to inoculum of the VAM endophyte Glomus caledonium was investigated. Infection began after a prolonged lag phase and remained at very low levels even after three months' growth. Neither very high rate~ of inoculum, nor very low P content of the soil raised the low infection level of the sulla plants. There appeared to be some differences in rate of infection among ten different ecotypes of sulla but the level of infection remained low in all cases. In all tested populations some plants remained uninfected. The low infection rate of sulla may therefore have a genetic basis. It was shown that the growth of H. coronarium is hardly improved by phosphate fertilization. This may explain the poor response of this plant species, adapted to grow in nutrient-deficient soil, to VAM. Programmes aimed at increasing the productivity in marginal soils through the introduction of efficient VAM endophytes should take into account the fact that certain plant species growing in marginal soils may not always benefit from mycorrhizal inoculation, due to their inherently low mycorrhizal dependency.

Introduction

Vesicular-arbuscular mycorrhizal (VAM) fungi generally act as plant-beneficial symbionts in soils deficient in available phosphate. For this reason they have been the subject of increasing interest in studies on the reclamation of nutrient-deficient soils for agriculture (Menge, 1983). Limitations to a massive introduction of VAM fungi in the field result on one hand from the difficulties of largescale production of inoculum, and on the other from their already widespread distribution; roots of most plant species are almost anywhere already colonized by mycorrhizal fungi, though not necessarily by the most efficient species. At present, the practical use of VAM fungi is limited to disturbed or fumigated soils where the VAM endophyte population is damaged. Field inoculation requires glasshouse selection of the most efficient fungal species in relation to spe-

213

cific host plant growing in a specific soil, assuming that the same growth responses will be reproduced in the field (Hayman, 1982; Abbott and Robson, 1982). Hedysarum coronarium L. (sulla) a leguminous plant typical of marginal soils in Central 'and Southern Italy but also growing widely in other areas of the Mediterranean basin, is known to play a key role on those soils which, due to their particular characteristics, are not suitable for other forage legume crops (Monotti, 1975). Previous studies on H. coronarium have shown that inoculation with Glomus caledonium, an endophyte known to promote growth in other cultivated plants (Schubert and Hayman, 1986), produces little infection and poor growth responses in this crop (Giovannetti and Hepper, 1985; Lioi and Giovannetti, 1987). Plant growth responses to VAM fungi are significantly influenced by factors such as host plant, endophyte, and soil (Bethenfalvay et al.,

214

Lioi and Giovannetti

1985). Therefore, we tried to determine the role of such factors for the relationship between H. coronarium, a plant species typically adapted to marginal soils, and the mycorrhizal endophyte G. caledonium. The purpose was to evaluate whether the low affinity between H. coronarium and G. caledonium can be improved. The following factors were studied: rate of infection, inoculum density, and efficiency of the endophyte; genetic variability, and response to phosphorus (P) of the host plant; very low P content of the soil. A highly mycotrophic plant species, Medicago sativa L. (lucerne), and a highly effective endophyte, G. monosporum, were used for comparing the influence of both plant species and fungal species.

Materials and methods

An alkaline sandy soil was collected near San Piero (Pisa) and, after sterilization for 1 h at 121~ to eliminate naturally occurring VAM endophytes, mixed with washed river sand (1:1 v/v). The soilsand mixture had the following characteristics: pH 8.1, 6 ppm NaHCO3 soluble P (Olsen analysis), 117 ppm available K, 0.73%o total N, 0.72% organic matter. Experiments were conducted in a glasshouse with temperatures ranging between 18 and 25~ and a daylength of 14h. The VAM fungi used as inocula, Glomus monosporum Gerd and Trappe, and Glomus caledonium Gerd and Trappe, were maintained on strawberry and onion stock plants respectively. Inoculum consisted of infested soil containing, as infective propagules, sporocarps, spores, root fragments, and external mycelium. To provide a common soil microflora, control plants received a filtrate from infested soil that had been passed through a sieve with a pore diameter of 50 #m. Seeds of H. coronarium (ecotype from Pisa) and M. sativa, were germinated in Petri dishes, selected for uniformity and transplanted into pots or vials arranged in randomized blocks. Shoot dry weights were obtained by drying for 48h at 91~ Roots were stained with trypan blue (Phillips and Hayman, 1970) and examined for mycorrhizal infection using the gridline intersect method (Giovannetti and Mosse, 1980).

Experiment 1

The first experiment was performed to evaluate the rate of infection, growth responses, and P uptake in sulla plants infected by G. caledonium. Lucerne and G. monosporum were used as plant and endophyte controls. Three seedlings were transplanted into each 0.81 volume pot, and inoculated by placing 3 g of crude inoculum near the roots of each plant, so each of them was provided with a sufficient number of fungal propagules for unlimited spread of infection (Giovannetti and Avio, 1985). Each treatment consisted of eighteen pots. Starting from the 15th day after transplanting, 9 plants from three pots were harvested every other week, shoot dry weights and % of root length infected were recorded. Shoots were analyzed monthly for P content by the phosphomolybdate method, and one-way analysis of variance was performed on the data obtained.

Experiment 2

Four inoculum rates were chosen to test whether higher inoculum doses increase G. caledonium infection on H. coronarium. Increasing quantities of crude inoculum (2.5, 5, 10, and 20 g) and a quantity of sterilized inoculum to make up 20 g were mixed with 480 g of experimental soil. Six replicate pots made up each treatment, and three sulla seedlings were transplanted into each pot. Six plants of M. sativa were inoculated at the rate of 5 g 9pot-~ to check the viability of the inoculum. Three harvests were performed at monthly intervals, washing and staining the whole root system of each plant to evaluate mycorrhizal infection.

Experiment 3

Ten ecotypes of sulla, originating from different areas of cultivation within the Mediterranean basin, were used to assess whether the low susceptibility of this crop to infection by G. caledonium is a character of the local sulla population or if it is present in other sulla ecotypes. Nine seedlings of each sulla ecotype, and nine seedlings of lucerne as control, were transplanted singly into vials contain-

VAM infection in Hedysarum coronarium 80

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b

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40

20

2(.

0

0

15

30

45

60

75

0

90

15

30

45

60

75

90

days Fig. 1. Progress in infection in a) H. coronarium and h) M. sativa by the mycorrhizal endophytes G. caledonium (A monosporum (0 0). Bars on data points indicate standard errors except when too low to graph.

ing 2 g of crude inoculum mixed with 28 g of the experimental soil. Six weeks after transplanting the plants were harvested, and their root systems were stained and assessed for mycorrhizal infection. Data on % of root length infected were analyzed by one-way analysis of variance after the necessary transformations.

Experiment 4 This trial was performed to evaluate the effects of increasing additions of P to the experimental soil on infection of H. coronarium and M. sativa by G. caledonium. White quartz sand (Sigma) was thoroughly mixed with increasing amounts of b

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115 3'0

4'5

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days Fig. 2. Shoot dry weights of a) H. coronarium and b) M. sativa uninoculated (Ha II) and inoculated with G. caledonium (A and G. monosporum (e O). Bars on data points represent standard errors except when too low to graph.

A)

216


Table 1. Shoot P contents of H. coronarium and M. sativa 30, 60, and 90 days after inoculation with G. caledonium and G. monosporum and of uninoculated control plants

Plant

Endophyte

Plant age (days) 30

60

90

P (%)

30

60

90

P (,ug. shoot - l )

H. coronarium

None G. caledonium G. monosporum

0.01 a 0.02a 0.06a

0.02a 0.02a 0.16b

0.03a 0.04a 0.22b

2.2 5.2 14.6

22.6 13.6 236.0

98.7 105.4 1013.0

M. sativa

None G. caledonium G. monosporum

0.08a 0.09a 0.08 a

0.09a 0.08a 0.16b

0.04a 0.09a 0.31 b

4.8 4.0 15.0

10.3 17.1 76.6

11.4 239.4 1398.0

Within plant species, data in columns followed by the same letter do not differ significantly from each other (p = 0.05 as determined by L.S.D.).

Ca(H2PO4) 2 to give final P concentrations of 0, 1, 2, 4, 8, 16, and 64ppm. Each P treatment was mixed with crude inoculum of G. caledonium to give a final quantity of 2 g in each vial. The P content of the O treatment was 0.4 ppm. Ten replicate vials each containing 30 g of sand were prepared for each P treatment. Two ml of nutrient Hoagland solution lacking P were added to each vial weekly. After 6 weeks, the plants were harvested, and the root systems were stained and checked for mycorrhizal infection.

Experiment 5

The responses of uninoculated sulla and lucerne plants to increasing P supply were tested in this trial performed as in experiment 4 except that the inoculum was sterilized before being added to the sand. After 6 weeks, plants were harvested and shoot dry weights recorded.

Results

The rate of infection in sulla roots by G. caledonium is shown in Fig la. This endophyte showed a very long lag phase--more than two months--before the first signs of penetration into sulla roots. This low aggressivity of G. caledonium was also found in the host plant lucerne, in which only 26% of root infection was reached after three months, a very low value for this highly mycotrophic plant species. G. monosporum, on the other hand, reached 10% and 21% infection in sulla and

lucerne roots respectively only 15 days after inoculation (Fig. 1). Inoculation with both fungi produced evident growth responses after about 50 days' growth (Fig. 2). Shoot dry weights of G. caledonium-infected sulla were slightly lower than those of the uninoculated plants. In lucerne, where G. caledonium consistently caused infection, data on shoot dry weights (Fig. 2b) and shoot P content (Table 1) showed the low efficiency of this endophyte, compared with G. monosporum, in the uptake or translocation of P to the host plant. Increasing rates of G. caledonium inoculum did not increase the poor infection of sulla plants (Table 2). At the lowest inoculum rate, G. caledonium failed to infect sulla roots, and at the highest inoculum rate (20g. pot-~), infection levels remained very low, showing no significant increase with time (Table 2). The poor ability of G. caledonium to infect sulla plants is confirmed from the data reported in Table 3. Ten ecotypes of H. coronarium, normally used for field cultivation in the respective areas of origin (Fig. 3), were screened for infection by G. Table 2. Effect of inoculum doses and time (days) on mycorrhizal infection by G. caledonium of H. coronarium roots

A m o u n t of inoculum ( g . pot -l ) 2.5 5 10 20

Root infection (%) 30 days

60 days

90 days

0 0 0 0

0 1" 4 1""

0 5 4 4

"" Two or more plants were not infected

V A M infection in Hedysarum coronarium Table 3. Infection by G. caledonium of ten different sulla ecotypes, expressed as percentage of root length infected and number of infected plants six weeks after inoculation

Reference number cf. Fig. 3

Origin area

1 2 3 4 5 6 7 8 9 10

Algeria Algeria Tunisia Italy Enna Italy Regio C. Italy Avellino Italy Roma Italy Grosseto Italy Pisa Italy Forli

Mycorrhizal infection (%)

No. of infected plants

2ab 1a 1a 0a 0a 3ab Iab 8b 2ab la

3 2 1 0 0 6 5 7 6 2

217

~4

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,~

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3. Origin areas of sulla ecotypes tested for infection by G. caledonium. For correspondence of reference number see Table 3.

Data followed by the same letter do not differ significantly from each other (p = 0.01 as determined by LS.D.)

caledonium. Six weeks after inoculation, none of the sulla ecotypes had reached the infection level present in lucerne roots (16%). Infection values in sulla roots ranged from 0% in ecotypes Enna and Reggio Calabria to 8% in the Grosseto ecotype. The presence o f some uninfected plants was a comm o n feature in all the ecotypes tested.

The inability of G. caledonium to infect sulla roots was not improved by very low P content in the soil (0.4 ppm); at this soil P content, lucerne had 12% of root length infected (Fig. 4). When not inoculated, the two plant species showed a different behaviour in response to phosphorus supply (Fig. 4b). The growth of sulla 60

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64 added

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i

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64

P (ppm)

Fig. 4. Effect of P content of soil an a) infection by G. caledonium of H. coronarium (grey columns) and M. sativa (open columns) and b) growth of the same plants uninoculated. Bars at the top of the columns indicate standard errors except when too low to graph.

218


plants was not increased by increasing soil P content and, even at the lowest P content (0.4 ppm), no deficiency symptoms appeared. By contrast, M. sativa showed an almost linear growth increase throughout the entire range of increasing soil P content, and at the lowest P dose plants were stunted though not necrotic.

Discussion Specific interactions between plant species and mycorrhizal fungi require the selection of an efficient VAM endophyte for each crop (Plenchette et aL, 1982). G. caledonium has recently been reported to be an effective endophyte, capable of exploiting very lowP conditions (Schubert and Hayman, 1986). When H. coronarium was used as host plant, G. caledonium showed a prolonged lag phase, poor infection and low efficiency in P uptake or translocation. All three factors may negatively influence the balance of the symbiosis, in that a prolonged lag phase may mean that some old roots are no longer susceptible to infection (Hepper, 1985), and at the same time that there is a lesser supply of P in the early stages of plant growth. Although plant growth responses are not directly related to the percentage of root infection, values recorded in sulla root systems were too low to contribute significantly to plant nutrition. Low P uptake or translocation efficiency, together with delayed root infection, may be responsable for the low shoot P contents found in G. caledonium infected plants. As regards the reclamation of marginal soils by means of H. coronarium, G. caledonium should be excluded from trials having this objective because, even at inoculum rate of 20g 9 pot ~ (equivalent to impractical doses of 120t. ha -x) and even after three months, sulla roots were only sparsely infected. High levels of root colonization have often been related to low soil P content (Crush and Caradus, 1980; Daniels Hetrick et al., 1984). Good results were in fact obtained with plants of lucerne grown in soil with very low P content and infected by G. caledonium. In sulla plants, however, the same low P concentrations did not stimulate infection as ex-

pected, and poor infection was obtained even at very low P contents, suggesting that phosphorus does not play a decisive role in initiating the sullaG. caledonium symbiosis. Local populations of sulla, a pioneer plant in marginal soils, are widely cultivated in Italy; they show considerable differences in behaviour and productivity because varietal selection had not yet reduced intraspecific genetic variability (Porceddu and Monotti, t976). The influence of genotype variations within a given species on the development and expression of a VA mycorrhiza has already been reported (Bertheau et al., 1980; Azcon and Ocampo, 1981; Krishna et aL, 1985). The ten sulla ecotypes we tested showed a certain degree of intraspecific variability in their susceptibility to infection by G. caledonium. Nevertheless the levels of infection in sulla populations are still significantly different from those achieved in lucerne at p = 0.01. This finding suggest that the resistance of sulla to infection by G. caledonium is at least partly controlled by the plant genome. Mycorrhizal H. coronarium plants gave poor growth responses even when infected with the highly efficient endophyte G. monosporum. The increase of P in the plant tissues produced only a small increase in shoot dry weights. This behaviour may be explained on the basis of the low P demand of this plant which, habitually growing in marginal soils, is probably adapted to live in the presence of very low quantities of phosphorus. Since the purpose if introducing efficient mycorrhizal fungi into marginal soils is to increase the yield of infected plants, programmes aimed at increasing productivity in these soils should take into account the fact that some pioneer plants such as sulla, adapted to growing in P-deficient soils, do not always benefit from VAM inoculation.

Acknowledgements Research work supported by CNR, Italy. Special grant IPRA-Subproject 1. Paper N 1076. The authors wish to thank M Macchia, University of Pisa, and M Monotti, University of Perugia, for providing the seeds.

V A M infection in H e d y s a r u m

coronarium

219

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