Suppression of mycorrhizal fungi in fescue by the acremonium coenophialum endophyte

Soil Bid. Biochem. Vol. 24, No. I. pp. 633-637, I!?92 Printed in Great Brilain. All rights reserved

003%0717/92$5.00+ 0.00 Copyright c 1992Pergamon Press Ltd

SUPPRESSION OF MYCORRHIZAL FUNGI IN FESCUE BY THE ACREMONIUM COENOPHIALUM ENDOPHYTE MYRA CHU-CHOU,~ B. Guo,’ Z.-Q. AN,’ J. W. HENDRIX,‘* R. S. FERRISS,’ M. R. SIEGEL,’ C. T. DOUGHERTY*and P. B. BURRUS* ‘Department of Plant Pathology and 2Department of Agronomy, University of Kentucky, Lexington, KY 40546, U.S.A. and ‘Forest Research Institute, Rotorua, New Zealand (Accepted IO February 1992) Summary-Propagule population densities of the nine mycorrhizal species detected in field plots planted to tall fescue free of the Acremonium coenophiulumendophyte were more than double those in plots planted to fescue heavily infected with Acremonium. Seedlings of Acremonium-free fescue detected more propagules

of most of the eleven mycorrhizal

species detected

in a field soil than seedlings

infected

with

Acremonium. Sporulation by three mycorrhizal isolates over 17 weeks was higher on fescue seedlings free of Acremonium than on seedlings infected with Acremonium. Since mycorrhizal fungi are believed to be confined to roots and Acremonium endophytes to shoots, the inhibitory effect of Acremonium on mycorrhizal fungi may be due to the translocation to roots of alkaloids produced by the AcremoniumFestuca symbiosis.

the cultivar Johnstone, a fescue-ryegrass hybrid which is free of Acremonium. Our objective here was to determine if A. coenophiulum inhibits G. macrocarpum and other mycorrhizal fungi.

INTRODUCTION Rotation of tobacco with tall fescue (Festuca arundinacea Schreb.) suppressed reproduction of the mycorrhizal fungus Glomus macrocarpum Tul. & Tul., the tobacco stunt pathogen (Modjo and Hendrix, 1986; Modjo et al., 1987), when tobacco was subsequently planted on the land (Hendrix et al., 1992). Suppression of this soilborne fungus is the reason fescue maintains productivity of soil for tobacco. However, fescue as a rotation crop for soybean was a good host for G. macrocarpum, as well as related species (An et al., 1990a). Tall fescue is often infected with a seed-transmitted endophytic fungus, Acremonium coenophialum Morgan-Jones & Gams (Clay, 1990; Siegel et al., 1987). The fungus is found in leaf sheaths and flowering stems but not in roots. Infected tissues contain toxic alkaloids that are deleterious to grazing animals. They also inhibit a variety of insects, confer resistance to rust disease (Ford and Kirkpatrick, 1989), and inhibit root pathogenic nematodes (Kimmons and Gwinn, 1990; Pedersen et al., 1988; West et al., 1988). In the tobacco experiment (Hendrix et al., 1992), Kentucky 31 tall fescue was used. Until the agricultural community became aware of the toxicity associated with Acremonium a few years ago, seed of Kentucky 31 were usually infected, and over 90% of fescue in Kentucky is infected (Siegel et al., 1984). In the soybean experiment (An et al., 1990a), we used

MATERIALS AND METHODS Acremonium effects on mycorrhizal propagules other soilborne microorganisms in the field

and

Field plots of fescue cultivars were planted in September 1986 in a randomized complete block design. Soil from plots of cv. Kentucky 31 either free of Acremonium or highly infected (87.5%) were sampled in April 1989. Presence of Acremonium was determined by enzyme-linked immunosorbent assay (Johnson et al., 1982) and by staining (Clark et aI., 1983). Plot size was 1.5 x 7.5m. Only blocks of soil harboring fescue (not weeds), ca 20 x 20 x 20cm. were dug up, then trimmed with a saw to cu. 15 x 15 x 15 cm. The top of the block containing crowns and leaves was removed. The soil with roots was mixed, and the two subsamples were cornposited. Propagules of mycorrhizal fungi were determined by the Most Probable Number procedure of An et al. (1990b), with Acremonium -free Kentucky 3 1 fescue seedlings as bioassay host, and identified according to Schenck and Perez (1990). For other fungi, a 50 g (fresh wt) sample was blended for 1 min, and a dilution series was prepared. The dilutions were plated on media selective for Pythium spp (Mircetich and Kraft, 1973), Rhizoctoniu spp (Sumner and Bell, 1982), Fusarium spp (Papavizas, 1967), and total fungi (Steiner and Watson, 1965). For nematodes, a

*Author for correspondence. 633

634

MYKA CHU-CHOL

50 g soil sample was screened through a 250 pm mesh sieve to remove debris, and nematodes were collected on a 44pm mesh sieve. Nematodes were separated by centrifugation in a sucrose solution as for mycorrhizal fungi and stored in deionized water for microscopic identification. Eflect

of Acremonium

of propagules

in fescue

of mycorrhizal

seedlings

fungi

on detection

in ,field soil

Efl‘ect

of

Acremonium of mycorrhizal

infection

of

.fescuc

Table I. Propagules of mycorrhual fungi, nematodes, and other so11 fungi detected in plots planted to fescue highly infected OTfree of the .Acremonium endoohvte NO

Mnoorgamsm

Propagules ~~ywrrhizul

fimg1

G. fecundl.spvr-urlr

43

24 + 7 17i_ II 12 f 7 1227 x*x 4+4 0 t1 0 7s T 32

IO’

I

33 + I4 27 i II 2’)+ IX l9i_4 15*4 8+8 4i4 4&4 IX1 f 46

G miuocurpum ti. constricturn G mm~.sporun, G. ~u.wiculurum G. rrfbrdum G nlOSSPUP

Total iVlWlUtd~_~ spp ~,~lrn~ hu.\ spp ffoplolaimus spp Protylmchus spp Non-parasitic spp Hrli~~ot~I
Other

P

’

(kg aoll)

spp

GIomus

G nmrrororpun,

0 33

0 44 0.25 0.21 0.0007 0 42 a 42 0 42 0.097

16i7 203 * 44 4+1 33* II 51 i I3

lil 104*x 4-tl 28 + 7 47 f 7

0.003 0.03 I 0.77 0.68 0.78

185 i I28 + I68 + 182 i

157+ II I55 i 59 244k56 148 * I3

0.26 061 0.22 0.26

soil /iingi

Fli.mrium

spp ( x

1II ‘)

spp ( x IO ‘) spp (x IO ‘) Total(xl0 6) Rhkortonia

Pythium

21 39 25 20

*Mean i SE Means of 4 replications

on

RESULTS

,fkgi

The influence of Acremonium on sporulation of three isolates of mycorrhizal fungi was determined using Acremonium-free and 100% infected Kentucky 31 seeds. Seeds were surface-sterilized with 0.5% sodium hypochlorite and germinated in the laboratory. The isolates were G. macrocarpum Perkins isolate, which caused tobacco stunt disease in Scott County, Kentucky (Modjo and Hendrix, 1986; Modjo et al., 1987); G. macrocarpum Schenck isolate, obtained from Abbott Research Center, which originally obtained it from N. C. Schenck in Florida; and G. mosseae, isolated from a soybean field in McLean County, Kentucky (An et al., 1990a, b). The spore population density of the inoculum was determined from subsamples of the inoculum sand-soil mixture, and viability of spores was determined (An and Hendrix, 1988). A IO-day old seedling was transplanted into a growth tube containing 50 g of an autoclaved mixture of equal parts of Maury silt loam topsoil and building sand on the bottom, a thin layer of soil inoculum containing about 25 viable spores, and a thin layer of sterilized soil-sand mixture on top. Because soil fertility influences sporulation (Kiernan et al., 1983), the experiment was conducted at a low rate of fertilization (1.1 g 1-i of 18-6-12 Osmocote slow release fertilizer, 18N-2.6P- 1OK, Sierra Chemical Co., Milpitas, Calif., U.S.A.) and at the manufacturer’s recommended rate (4.5 g 1-l). Seedlings were grown in a greenhouse. The spores on the roots and sieved from the soil from four plants from each treatment were determined periodically. The presence or absence of Acremonium was determined by staining.

High endophyte

endophyte

G. uJncrdm1.w

A dense Kentucky 31 fescue sod heavily infected with Acremonium was sampled 1 June 1990, 3 weeks after plowing. Soil samples were composites of twenty 15cm deep cores 2.5 cm dia, taken randomly from each of 12 plots 5 x 30 m in width and length. Mycorrhizal propagules were bioassayed and identified as above. The seed used for bioassay were fescue 92% infected with Acremonium (Johnson et al., 1982) harvested the previous spring from the site of the soil samples for this study; fescue free of Acremonium harvested the previous spring from an adjacent site; and pearl millet (Pennisetum americanum Leeke cv. 3-MIL-X Hybrid). The two fescue seed lots are isolines traced back to the original Kentucky 31 source.

sporulation

et (11.

Acremonium

eflects

other soilborne

on mycorrhizal

microorganisms

propagules

and

in the ,field

Populations of propagules of all mycorrhizal species and of total mycorrhizal propagules were lower in plots planted to Acremonium -infected fescue than in plots planted to Acremonium-free fescue (Table 1). The variability of the population data was high, which may be the reason the differences usually were not significant statistically; however, a trend is evident. Populations of other microorganisms were also affected (Table 1). Populations of the parasitic

Table 2. Effect of A. coenophiulum infection of fescue seedlings used as assay hosts on populations of mycorrhizal fungi detected in a soil sampled 3 weeks after plowing a fescue sod heavily infected with A. coenoohiafum

Pearl millet

Species

Endophytefree fescue

Propagules

G.pallidurn

518 + 122a’ 281 li_ 83ab 316+65a

G. oersicu&rum G. microcarpum G. culedonium

90 f

l2a

G. efun,ca,um G. constricturn

5b 22 * 7a 21 k 6ab

Gigaspora

21 _t5a

Entrophospora

i@-eyuens

albidu

G. leptotichum G. aggrqatum Gigaspora

gigantea

Scuteilospora G. macrocarpum

Total

fulgida

24 _t

’ _______

(kg soil)

434 i: 6lab 386 i 68a 224 f 43ab 42 +_ 9b 55 * l3a lZf4ab 32 f 7a 6 + 3b

8 + 4a

5 * 3a

4 i_ 2a 2* la

2 k 2ab 5 _t 3a

Oa Oa

1307 k 217a

4 f

Endophyteinfected fescue

229 206 154 40 32

i40b + 42b ? 29b + 9b k l2ab Ob 182 7b 8 + 5b I * la Ob 3 _t 2a OZi

2a

I _t la 1208 + 145a

I + la 691 + 86b

*Mean f SE. Means of 12 replications. Means followed letter within a line are not different (LSD, P = 0.05)

by same

Mycorrhizal fungi and fescue Acremonium endophyte

635

Effect of Acremonium infection sporulation of mycorrhizal fungi

nematodes Helicotylenchus spp and Tylenchus spp were suppressed in plots of fescue infected with Acremonium, while those of Hopiolaimus spp, nematodes Pratylenchus spp, and non-parasitic were not significantly affected. Acremonium appeared to suppress populations of total fungi and Fusarium spp and favor those of Rhizoctonia spp and Pythium spp, but none of these effects were significant statistically.

of fescue

Although sporulation was variable, Acremoniumfree fescue seedlings usually supported higher reproduction than Acremonium-infected seedlings (Fig. 1). An exception was seedlings inoculated with the Schenck isolate of G. macrocarpum and fertilized with the higher rate of fertilizer. The higher rate of fertilizer had no effect on sporulation of this isolate on Acremonium-infected seedlings, but sporulation on Acremonium-free seedlings was suppressed to a level similar to that on the Acremonium -infected seedlings. Over all sampling dates, sporulation on Acremoniumfree seedlings was usually statistically higher than that on Acremonium-infected seedlings, with the exception of the Schenck isolate of G. macrocarpum at the higher fertilizer rate (Table 3). Regardless of the presence of the Acremonium endophyte, tall fescue did not appear to be a good host for two of the three isolates. Sporulation by

Effect of Acremonium in fescue seedlings on detection of propagules of mycorrhizal fungi in field soil

Lower populations of most mycorrhizal species were detected with Acremonium -infected fescue seedlings than with seedlings free of Acremonium (Table 2); with two of them, and with totals of all mycorrhizal fungi, the differences were statistically different. Similar populations were detected with Acremonium -free fescue and pearl millet. Low fertilizer

Normal fertilizer

0 Acremonium free l

Acremonium infected

G. mosseae

250

on

G. macrocarpum (S)

-I 19

FfyPq, 5

7

9

11

13

15

17

19

Time (weeks)

Fig. I. Spore production by three isolates of mycorrhizal fungi on fescue plants either free of or infected with the A. coenophialurnendophyte over a I7-week period, as influenced by fertilizer rate. G. mucrocurpum (P) = the Perkins isolate from Kentucky; G. mucrocarpum (S) = the Schenck isolate from Florida. Bracket indicates standard error; if not visible, the standard error was small. *Indicates means for that

date statistically different (t-test, P = 0.05).

MYRA CHU-CHOU

636

et al.

Table 3. Effect of the A. coenophinlumendophyte on sporulation of

three mycorrhizal mlates rmts of fescue seedlings grown at two fertilizer levels and sampled Over a 17.week period Low fertilizer NO endophyte

lsolare

Propagules G. m”SScae G. macrocarpum Perkins isolate Schenck isolate

21*3* 25 * 3 18Ok28

Endophyte

Normal fertilizer P

’

NO endophyte

l5i2

0.098

17*3

I3 * 2 64+ IO

0.0036 0.0003

24 i 3 935 IO

15 & 2 90112

Propagules

P

Endophyte ’ __~~ IO? I

plaw

on

plant

0.029 0.03x 0.88

‘Mean _+SE.

the G. mosseae isolate and the Perkins isolate of G. macrocarpum was usually lower than the number of viable spores used as inoculum (Fig. 1, Table 3). Sporulation by these isolates, although sparse, occurred, however, for spores were consistently observed still attached to roots in groups, not characteristic of infecting spores still attached to roots by infecting hyphae. DISCUSSION

These

experiments measured suppression by of different variables of the biology of mycorrhizal fungi: populations in the field, an influence on populations determined with bioassay seedlings varying in Acremonium colonization, and sporulation. The suppressive effect appears to be on mycorrhizal fungi in general, not just on certain species. Acremonium present in fescue plants in a field sod did not appear to select propagules of mycorrhizal fungi tolerant of Acremonium or its toxic alkaloids. Suppressive effects of Acremonium or its metabolites on insects (Cheplick and Clay, 1988; Johnson et al., 1985) or nematodes (Kimmons and Gwinn, 1990; Pedersen et al., 1988) often is not absolute. When not absolute, the suppression may be evident but not statistically significant. While effects on populations of all mycorrhizal propagules were significant (Tables 1 and 2), those on many individual species were not. Such data are highly variable, and less than absolute inhibition by Acremonium may introduce further variability. While Acremonium endophytes are believed to occur in leaf sheaths and reproductive structures, but not roots, mycorrhizal fungi and nematodes confined to roots are affected by Acremonium. Transport of toxic metabolites may be the reason. Three types of toxic alkaloids have been identified in Acremonium infected fescue (Siegel et al., 1990). The azaindolizine, peramine, produced by many endophytes (Rowan er al., 1986; Siegel et al., 1990), was found in all plant parts including roots (Fannin et al., 1990). Lolines, the saturated pyrrolizidine alkaloids typical of tall fescue-A. coenophialum associations and which accumulate up to 0.8% dry weight (Kennedy and Bush, 1983; Belesky et al., 1987), were found in fescue roots, although in lower concentrations than in above-ground tissues (Bush et al., 1992). Lolines are Acremonium

potent insecticides (Johnson et al., 1985; Yates et al.. 1989). Proof of toxicity of metabolites to Glomales mycorrhizal fungi will be difficult to obtain because of the obligately parasitic nature of these fungi. While our observations are limited, it appears that the influence of Acremonium below ground may be confined to the roots, but not extraradical areas of the rhizosphere. Parasitic nematodes were affected by Acremonium, but non-parasitic nematodes were not. Propagules of most of the non-mycorrhizal fungi enumerated probably were extraradical, especially those present in high populations (“total” fungi and species of Fusarium and Pythium). Sporulation by two of the three mycorrhizal isolates studied appeared unaffected by the fertilizer rates used. Sporulation by the Schenck isolate of G. macrocarpum, however, was reduced at the higher fertilizer rate on Acremonium endophyte-free seedlings, but not on Acremonium -infected seedlings. This isolate had much higher sporulation potential than either of the other two. Realization of maximum potential to sporulate may be inhibited by such disparate influences as Acremonium or high fertilization, but a base level of sporulation may be resistant to either influence or both together. While the suppressive effect of Acremonium on mycorrhizal fungi is apparent, mycorrhizal isolates may vary in efficiency to colonize and sporulate on fescue, whether or not Acremonium is present. The two isolates of G. macrocarpum we studied varied in this respect (Fig. 1, Table 3). Inherent capacity to colonize fescue may be more important than presence of Acremonium in the disparate results of the western Kentucky soybean experiment (An rr al., 1990a) and the central Kentucky tobacco experiment (Hendrix et al., 1992). Acknowledgements-We thank Janet Finley, Chris Hoskins and Walter Ho&n for assistance.

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