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Gigaspora gigantea: parasitism of spores by fungi and actinomycetes
458
Mycol. Res. 98 (4): 458-466 (1994) Printed in Great Britain
Gigaspova gigantea: parasitism of spores by fungi and actinomycetes
PAU-JU LEE' A N D R.E. KOSKEY Department of Botany, University of Rhode Island, Kingston, Rhode Island 02881, U.S.A.
Fungi and actinomycetes were isolated monthly for one year from spores of the arbuscular mycorrhizal fungus Gigaspora gigantea recovered from a maritime sand dune. Spores in four different stages of vigour (newly formed, greenish-yellow healthy; yellow, moribund, mottled with brown spots; brown; and dead, blackened and collapsed) were used for isolation. From 272 isolates cultured from crushed, surface-disinfected G. gigantea spores, 44 species of fungi and six actinomycete species were recovered. The five most frequently isolated organisms were Acremoniurn sp., Chrysosporium paruum, Exophiala werneckii, Trichoderma sp. and Verticillium sp. The species lists derived from the four spore types differed significantly. Thirty-one of the isolated species were tested For their ability to parasitise healthy G. gigantea spores and to invade spores killed by a hot water treatment. Twenty-two species could function as pathogens, forming intemal projections (IP), fine radial canals (FRC) or both in the spore wall. IP were induced by Acremoniurn sp., Chysosporium parvum, Cladosporium sp., Geomyces pannorum, Oidiodendron sp., Sporothrix sp., Verticillium sp. and by the actinomycetes Nocardia sp. and Streptomyces sp. The IP length was positively correlated with duration of exposure to the parasites. In the bioassay, both IP and FRC were formed by live spores, while heat-killed spores possessed only FRC after penetration by the test microorganisms. Pathogenicity differed among the parasites and was greatest for Verticillium and Acremoniurn.
Observations suggest that parasitic fungi and actinomycetes may significantly reduce populations of spores of arbuscular mycorrhizal fungi (AMF) in the field and induce adverse effects on mycorrhizally dependent plants (Ross & Ruttencutter, 1977; Daniels & Menge, 1980; Ross & Daniels, 1982; Paulitz & Menge, 1986). However, despite the potentially great importance of such parasites, only five species of microorganisms (all of them fungi) have been shown to be capable of parasitizing spores of AMF. These include Anguillospora pseudolongissima Ranzoni (Daniels & Menge, 1980; Paulitz & Menge, 1986), Hurnicola fuscoafra Traaen (Daniels & Menge, 1980), Sfachybotrys chartarum (Ehrenb. ex Link) S. Hughes (Siqueira ef al., 1984), and two chytridiaceous species R h i z i d i o m y ~ o ~ s isfomafosa s Sparrow (Schenck & Nicolson, 1977; Sparrow, 1977) and Spizellomyces acuminafus (D. J.S. Barr) D. J. S. Barr (Barr, 1984). Two other species, Phlycfochyfrium kneipii Gaertn. (Tzean & Chu, 1985) and P. plurigibbosurn D. J. S. Barr (Tzean, Chu & Su, 1983), were reported as pathogenic to spores of AMF, but these claims appear to be based on misidentifications of S. acuminafus (Barr, pers. communication). A few other species of fungi and actinomycetes have been isolated from obviously parasitised spores of AMF and are suspected as parasites, but their ability to parasitise healthy
Current address: Department of Mathematics and Science Education, Tainan Teachers College, Tainan, Taiwan, R.O.C. * Corresponding author.
spores has not been proven experimentally. These include S. puncfafum (Koch) D. J . S. Barr ( = Phlycfochytrium puncfafum
Koch) (Ross & Ruttencutter, 1977; Daniels, 1981; Paulitz & Menge, 1984), an unidentified 'Pyfhium-like' species (Ross & Ruttencutter, 1977), a species of Cephalosporiurn (Malencon, 1947), and various unidentified fungi and actinomycetes (Mosse, 1956; Petitberghein, 1956; Godfrey, 1957). All of the parasites or suspected parasites listed above were isolated from obviously parasitised AMF spores, often from spores recovered from greenhouse-grown pot-cultures rather than from natural conditions. There has been no systematic isolation from healthy, aged, moribund, and dead spores of any AMF using material collected from the field. Species of potentially important parasitic micro-organisms may have been overlooked because of emphasis on a single category (parasitised) of spores. Two distinct morphological features of parasitised spores and hyphae of AMF have been described and thought to be induced during the penetration of the fungal wall. In one, it is presumed that the parasites create narrow channels (fine radial canals (FRC) [Petitberghein, 1956; Godfrey, 19571) in the wall of the AMF. In the second type, conical or digitate structures (internal projections (IP) [Mosse, 1956; Petitberghein, 1956; Godfrey, 1957; Mosse & Bowen, 19681) form on the inside of the spore walls where penetration occurs. These features have been observed in spores of AMF for over 80 years (Bucholtz, 1912), and the correlation between parasitism and FRC and IP was made by Mosse (1956),
Pau-Ju Lee and R. E. Koske Petitberghein (1956) and Godfrey (1957). Just three species (Rhizidiomycopsis stomafosa, Spizellomyces punctatum, and S. acuminatus [as P. kneipii]) have been shown to be capable of inducing FRC (Ross & Ruttencutter, 1977; Schenck & Nicolson, 1977; Sparrow, 1977; Tzean, Chu & Su, 1983), and only S. acuminatu (as P. plurigibbosurn) has been experimentally shown to induce IP (Tzean & Chu, 1985). The purposes of the present study were to determine which fungi and actinomycetes were intimately associated in the field with spores of Gigaspora gigantea (T. H.Nico1 & Gerd.) Gerd. & Trappe and to screen some of those isolates for pathogenicity to AMF spores. Gigaspora giganfea was selected for study because it is one of the dominant AMF species in coastal dunes of the U.S. Atlantic seaboard (e.g. Koske & Halvorson, 1981; Koske, 1987; Gemma, Koske & Carreiro, 1989). A further aim was to assess the relationship between parasitism and the presence of FRC and IP in spores.
MATERIALS A N D M E T H O D S Monthly isolation of micro-organisms from four stages of spores
Fungi and actinomycetes were isolated from spores of G. giganfea in four different stages of health from monthly collections (Mar. 1989 to Feb. 1990) from the barrier sand dune at Moonstone Beach, Rhode Island (RI). The collection area had a dense cover of American beachgrass (Ammophila breviligulafa Fern.). A stratified random sampling technique was used to collect 15 samples each month. Spores were sampled extracted and classified mostly by colour into healthy, moribund and dead spores (Lee & Koske, 1994). Moribund spores were further differentiated into mottled and completely brown categories, resulting in four life stages healthy, mottled, brown and dead. Each month 20 spores of each stage were surface-disinfected in 2 % Clorox@(0.105 % NaOCl,) (Koske, 1981) for 2-3 min followed by three rinses in sterile deionized water. To ensure that micro-organisms that were potentially pathogenic to spores of G. giganfea were isolated, only surface-disinfected spores were used. The 20 surface-disinfected spores of each stage were crushed in 1ml of half-strength MYP solution (Difco Malt Extract, 7.0 g ; Difco Soytone, 1.0 g ; Difco Yeast Extract, 0.5 g ; distilled water, 1 1) (Bandoni, 1972) to which 50 mg 1-' tetracycline (MYPT) were added to suppress bacterial growth. A 0.2 ml portion of the crushed-spore suspension in MYPT was spread evenly in each of five 100 x 15 mm Petri dishes containing 20 ml of MYPT agar (MYPT 1.5 % Difco Bacto agar). Plates were incubated for up to two weeks at room temperature (21-27 OC) and examined daily for developing colonies. Results were recorded as the number of colonies per plate. From each colony a culture was made for identification and subculturing. Identifications were made on MYPT agar by the agar slide method (Koch, 1972). Actinomycetes and hyphomycetes were identified only to genus (Barron, 1968; Campbell & Stewart, 1980; Carmichael ef al., 1980). Permanent slides were prepared by mounting isolated micro-organisms in a polyvinyl alcohol-lactic acid-glycerol solution (PVLG)
+
459 (Omar, Bolland & Heather, 1979) containing 0.5 % cotton blue. Voucher specimens are lodged in the collection of the first author. Additional healthy spores were isolated monthly from the dune and used for pathogenesis assays (see below). Attempts were made to isolate chytrids by placing brown spores in Petri dishes containing autoclaved tap water or pond water baited with pine pollen or other natural substrates such as snake skin, grass straw, or surface-disinfested spores of G. giganfea. Plates were incubated in a refrigerator (4') (Barr, 1987).
Data analysis
A modified Sorensons' Coefficient of Similarity (Bray & Curtis, 1957; Southwood, 1978) was calculated to compare the populations of micro-organisms isolated from spores in different stages of health. The calculations considered both presence and abundance of each species. Abundance was determined by measuring the number of colonies of each species that developed on the isolation plates. Similarity was calculated by the formula:
where aN = the total number of colonies developing on the 5 Petri dishes plated with spore suspension from spores in stage 'a' (i.e. healthy, mottled, brown, or dead), and bN is the total number of colonies from spores in stage 'b' and cN is the number of colonies in common.
Pathogenicity testing of isolated micro-organisms
In this study, the term pathogenicity is defined as the successful establishment of a micro-organism thallus on or in the living spore of G. giganfea as shown by the formation of IP, FRC, or both in the spore wall. Micro-organisms able to colonize live spores were thus regarded as pathogens. Of the 50 species of micro-organisms isolated from spores of G. gigantea, 31 species (including two isolates of one species, Geomyces pannorum) were selected for pathogenicity testing (Table 1).Micro-organisms were initially sorted into three groups on the basis of hyphal diameter: < 1 pm, 1-2 pm, and > 2 pm. Preliminary observations of the diameter of the FRC and the canals of IP of parasitised spores, indicated that most pathogenic isolates would fall into the first two categories. Greenish-yellow, healthy spores of G. gigantea, previously surface-disinfected with 0.105 % NaOCl,, were exposed to active cultures of the test organisms to assess their pathogenicity. The spores were freshly isolated from a sand dune soil (Moonstone Beach, RI) by wet-sieving and were exposed to a brief heat treatment before use in the pathogenicity test. In preliminary tests, untreated spores were slow to become parasitised, although later tests showed otherwise, and a heat treatment (immersion in a water bath at 35' for 5 min) which caused little loss in viability (Lee, 1991) was performed to make them more susceptible to parasitism. For the pathogenicity assay, a 100 x 20 mm Petri dish containing 20 ml of water agar (Difco Bacto agar, 15 g ;
Parasitism of spores of Gigaspora gigantea Table 1. Micro-organisms isolated from spores of Gigaspora gigantea
Stagesb
NO.^
*mycelia sterilia I 'mycelia sterilia I1
B, D M, 8, D
4
Actinomycetes 'Nocardia sp. 'Streptomyces sp. I *Streptornyces sp. I1 'Sfreptomyces sp. I11 'unidentified sp. I 'unidentified sp. 11
M M, 8, D M M H, M, B, D B
1 5 1 1
Av. colsd
Fungi 'Acremonium sp. *Arthrinium phaeospermum Aspergillus flawus Aspergillus nidulans Aspergillus terrew Aspergillus versicolor *Cercosporella persicae Chaetomium sp. 'Chysosporium parvum 'Chdosporium sp. Cylindrocarpon sp. "Epicoccum sp. *Exophiah werneckii Fonseceae pedrosoi 'Fusarium sp. I Fusarium sp. I1 Fusicladium sp. Fusidium sp. *Geomyces pannorum I "Geomyces pannorum I1 'Geotrichum candidum 'Gliomastix murorum "Gliomastix sp. 'Humicola fuscoatra Hyalodendron sp. "Isaria sp. I Isaria sp. I1 Monacrosporium sp. Monilia sp. 'Mortierella ramanniana 'Oidiodendron sp. Papulospora sepedonioides Penicillium sp. 'Phialophora sp. Piedraia hortae Septonema sp. *Spicaria lilacinum 'Sporothrix sp. 'Sporothrix sp. I1 Stemphylium sp. 'Trichoderma sp. "Tripospermum sp. *Verticillium sp. I Verticillium sp. I1 7.3 f16.1 2.8 f8.3
2
0-lf0.3 0.6f 0.8 0.1k0.3 0.1 k 0.3 0.4f0.7 0.1 f0.3
4
1
* indicates species or isolate used
in pathogenicity tests. Stage of spores of Gigaspora gigantea from which the listed microorganisms were recovered. H, healthy; M, mottled; B, brown; D, dead. Number out of 12 months sampled in which the microorganism was isolated. Average number of colonies isolated per month s . ~ .
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deionized water, 1 litre) was inoculated with a 6 rnrn diameter disk cut from the margin of a 7-day-old colony of the test organism growth on MYP agar. Ten to 20 surface-disinfected, yellow (therefore assumed not parasitised) spores of G. gigantea were placed on the water agar surface 5-10 mm from the edge of the inoculum disk. As controls, spores were arranged around a disk of sterile MYP agar. Each treatment was replicated three or more times. Plates were incubated at room temperature (21-27O) for three months, when spores were recovered and mounted in PVLG with or without 0.05 % cotton blue and examined microscopically. Parasitic potential of micro-organisms was assessed by counting the number of FRC or the number and length (vm) of IP that occurred on each spore. The 11 most pathogenic species in the above experiments were re-tested using the same bioassay, each using 10-20 spores replicated three times. Petri plates were sampled after 0.5 to 3 months, depending on the rapidity of mottling and browning, and the percentage of spores with IP or FRC was determined. Preliminary pathogenicity assays on water agar were slow, so a similar experiment was conducted (after Daniels & Menge, 1980) with a few species of micro-organisms in autoclaved dune soil. Dune sand from Moonstone Beach was passed through a 1.5 mm sieve, washed in tap water, air dried, and autoclaved for 1h at lZ1° on two successive days. Fifteen to 20 heat-treated, surface-disinfested yellow spores were placed on a sterile filter paper (5.5 cm diam.). The filter paper was then folded in half and set in a 80 x 100 mm culture dish containing ca 250 ml of the twice-autoclaved sand (moisture content ca 20%). Five agar plugs (8-10 mm diam.) from the margin of actively growing colonies of Streptomyces sp., Acremonium sp., Chysosporium sp., and Verticilliurn sp. on MYP agar were placed on the filter paper and covered with moist, sterile sand to a depth of 10 mm. The dishes were covered, and the culture incubated at room temperature (21-27'). Controls were identical except that agar plugs were not used. Each treatment was replicated three times. Spores were removed, stained, mounted, and examined intermittently as in the previous experiment to determine parasitic infection.
Re-isolation of pathogenic micro-organisms from spores used in the bioassay To determine if the test micro-organisms had penetrated the healthy spores of G. gigantea in the pathogenicity bioassays, one or two spores from each treatment were removed from the assay plate after 3 to 4 months, surface-disinfected, and placed on MYP agar. The identity of the micro-organisms that grew from each parasitised spore was confirmed.
Time-course and heat-treatment experiment
An isolate of Verticillium previously shown to be a vigorous pathogen of spores of G. giganfea was used to determine the rate at which spores were parasitised. Assay plates of water agar were set up as previously described using heat-treated and non heat-treated spores. Spores were sampled after incubation for 1, 2, 4, 6, 7 and 9 wk. The number and length
Pau-Ju Lee and R. E. Koske of IP formed in the spores was assessed. Sixteen to 30 spores were examined for each treatment and sampling time. The variation in number of spores assessed resulted from the browning of some spores in the assay plates following surface-disinfestation.These spores appeared healthy (yellow) after exposure to the sterilizing treatment, but later (after the assay plates had been set up) turned brown. Such spores were excluded from assessment. Data were analysed for significant differences in pathogenicity between micro-organisms using one way analysis of variance (ANOVA). Response of live and dead spores
From the morphology of the IP in spores of G. giganfea it can be suggested that the structure of the IP is built up by the fungus responding to attempted invasion by a parasite. To test whether a living spore was required to form IP, both live and dead spores were placed in agar assay plates with two known IP-inducing parasites, Verticillium sp. and Acremonium sp. Spores for testing were killed by placing them in boiling water for 5-10 min until they turned brown. Boiled spores did not germinate. Spores were incubated with the test fungi for up to 4 months, stained with cotton blue or trypan blue in PVLG, and examined.
RESULTS Monthly isolation of micro-organisms
Initially, 272 isolates were cultured from the four stages of spores collected throughout the year. The 272 isolates represented fifty species of hyphomycetes and actinomycetes (Table I).The most frequently isolated species were Exophiala werneckii, Acremonium sp., Verficillium sp. I, Trichoderma sp. and Chysosporium parvum. The four most frequently isolated species also were the species that formed the most colonies in the dilution-isolation plates. The number of species associated with spores of G. gigantea increased with the age of the spore. Healthy spores had the fewest associated species (8) and dead spores the most (31). Seventeen species were isolated from mottled spores, and 24 species from brown spores. Significant differences occurred among the microfloras derived from the four different stages of spores (Table 2). Greatest similarity was found between the associated microfloras of healthy and mottled spores (S = 0-58). Brown and dead spores also showed a comparable similarity coefficient (S = 0.52). Least similarity occurred between the inhabitants of healthy and dead spores (S = 0.0007). The species isolated from dead spores differed most from those obtained from other stages. Only three species were isolated exclusively from healthy spores (Aspergillusflavus,A. versicolor, and Isaria sp. I [Table I). Ten of the 31 species isolated from dead spores were not isolated from other spore stages
Pathogenicity testing of isolated micro-organisms
There were no differences between results obtained with assays on water agar or in soil, and only results from the water agar tests are presented here. Of 32 isolates tested ( = 31 Table 2. Similarity between fungal popidations isolated from surfacedisinfected spores of Gigaspora gigantea in four stages of health using modified Simpson's Coefficient of Similarity'
Stage Stage
Healthy
Mottled Brown Dead
< 0.01
0.58
Mottled
Brown
-
-
0.11 0.08
< 0.01
0.52
a Calculation is based on presence and abundance of micro-organisms derived from spores. See text.
Table 3. Pathogenicity of micro-organisms isolated from spores of Gigaspora gigantea
EffectC
Fungi Acremonium sp. Arthrinium phaeospemum Cercosporella persicae Chysosporium parvum Chdosporium sp. Epicoccum sp. Erophiala werneckii Fusarium sp. I Geomyces pannorum I Geomyces pannorum I1 Geotrichum candidum Gliomastix murorum Gliomastix sp. Humicola fuscoatra Isaria sp. I Mortierella ramanniana Oidiodendron sp. Phialophora sp. Spicaria lilacinum Sporothrix sp. Sporothrix sp. I1 Trichoderma sp. Tripospemum sp. Verticillium sp. I
mycelia sterilia I mycelia sterilia I1 Adinomycetes Nocardia sp. Streptomyces sp. I Streptomyces sp. I1 Streptomyces sp. 111 unidentified sp. I unidentified sp. I1
Xu
Y"
IP
FRC
IH
1-2
M D M M B
+ + + + +
+
+ + +
>2 >2 1-2 > 2 > >2 >2 1-2 1-2 >2 1-2 1-2 >2 1-2 >2 1-2 1-2 >2 1-2 1-2 >2 >2 1-2
< <
-
-
+ + +
-
-
+ +
+ -
-
-
D
-
-
-
D H B H M H M M B M B
-
-
-
-
-
-
-
2
D
B B B
H D
+ + + + + -
-
+
+ +
B M l l
M M
<1
M
<1 <1 <1
M
B B
+
+
M
>2 1-2
-
+ +
+
+
-
+
+
-
+ + -
+ +
+
+
+
-
-
+ +
+
+
-
+
-
+
+
+ +
-
-
-
-
-
-
-
Diam. (w)of hyphae on MYP. Stage of spores of Gigaspora gigantea from which the listed microorganisms were recovered. H, health; M, mottled; B, brown; D, dead. Formation of internal projections (IP), fine radial canal (FRC) and internal hyphae (IH) in healthy, surface-disinfested spores of Gigaspora gigantea exposed in agar culture (see text) to the test organisms for 3 months. ' ' indicates presence, ' - ' indicates absence. a
Isolation of chyfridiaceous fungi
Attempts to isolate chytridiaceous fungi failed. Zoosporangia were not produced on any autoclaved natural material or on surface-sterilized spores.
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Parasitism of spores of Gigaspom gigantea
F i g s 1-2. Parasitism of spores of Gigaspora gigantea by Streptomyces sp. I. Fig. 1. Fine radial canals (FRC) in face view are visible (small arrows) as are internal projections (IP) (large arrows). Bar = 50 wm. Fig. 2. Side view of fine radial canals (arrow)in wall. Interior of spore is to the right of the wall. Bar = 20 urn.
species), 23 (72%) were capable of functioning as pathogens of spores of G. giganfea (Table 3). Eleven isolates, including such common soil species or genera as Acremonium sp. Ckysosporium parvum, Cladosporium sp., Geomyces pannorum, Sporofhrix sp., Sfrepfomyces sp. I, and Verticillium sp. induced both IP and FRC (Figs 1-8). Species forming only FRC included Exopkiala werneckii and Humicola fuscoafra. Internal projections only (no FRC) were induced by ten species including Epicoccum, Geotrichum candidum, Isaria, Oidiodendron, and four species of actinomycetes. No IP, FRC or internal hyphae were formed in the spores in the controls or in spores exposed to Fusarium sp., Gliomastix sp., Mortierella mmanniana, Pkialophora sp., Sfreptomyces sp. 111, Trickoderma sp., Tripospermum sp., and two actinomycetes. Hyphal diameter on agar did not differ significantly between parasitic and non-parasitic isolates or species, although the average diameter of hyphae of parasitic isolates was slightly narrower (av. 1.64 Vm v. 1.80 ym). Of the 31 species tested for pathogenicity, those originally isolated from healthy or mottled spores more frequently were pathogenic than were species isolated from brown or dead spores (Table 4). Eighty-two per cent of species that were isolated from healthy or mottled spores were pathogenic in the bioassay (Table 4), while only 60% of the isolates from brown and dead spores were pathogenic. Species capable of inducing IP were isolated from healthy, mottled, brown and dead spores. Eleven species were assessed for their ability to parasitise healthy spores of G . giganfea over a period of two weeks to three months (Table 5). After 2 wk, 53-85% of the spores exposed to Acremonium and Verticillium were parasitised. B y three months, eight of the 11 species had parasitised 50% or more of the spores. Species were ranked by the number of IP that they induced
Figs 3-4. Parasitism of spores of Gigaspora gigantea by Verticillium sp. I. Internal projections (IP) formed in response to attempted penetration of the spore wall by the parasite. Fig. 3. Numerous
worm-like IP (Black arrow on white background) are present. Small, wart-like structures on the inner surface of the spore wall are not the result of parasitism. Bulbous base is indicated (black arrow). Bar = 30 wm. Fig. 4. Long IP typically induced by Verticillium. Bar = 10 urn.
after a 3- or 4-month incubation period (Table 6). As in the parasitism study, Verticillium and Acremonium were the most aggressive pathogens. Verticillium induced an average of 47.1 IP/spore after three months, and some spores had more than 100 IP. The average size of the IP induced by Verticillium was 4 x 120 Vm. Germ-tubes of G . giganfea also were attacked by hyphae of Verticillium (Fig. 9), and IP were formed (Fig. 9). In spores in which IP were formed, the first visible sign of parasitism was the occurrence of brown spots on the spore wall, resulting in a mottled appearance to the spore. IP developed first from the inner, germinal wall of the spores as the host deposited layers of cell wall-like material in front of the advancing penetration hypha. The material that was deposited appeared similar in composition to that of the laminated spore wall. The sequential deposition of materials at the point of attack was apparent as concentric layers that were especially evident at the tip of the IP (Fig. 8). Each IP enclosed one or two fine channels ( < 1 ym diam.) in which the parasite occurred (Figs. 3-8). Canals were branched or unbranched, and some IP included more than one canal (Fig, 7). The hyphae
Pau-Ju Lee and R. E. Koske
463 Table 5. Percentage of spores of Gigaspora gigantea parasitised by selected micro-organisms on water agar Culture time (months)
Acremonium sp. Verticillium sp. I Chysosporium parvum Actinomycete I Sporothrix sp. I Streptomyces sp. Nocardia sp. Geomyces pannorum I Streptomyces sp. I1 Cladosporium sp. Sporothrix sp. I1
n.d. n.d.
n.d. n.d.
n.d. n.d.
40 41
* n.d., not determined.
Table 6. Formation of internal projections {IP) by healthy spores of Gigaspora gigantea in response to selected micro-organisms after 3 or 4 month's incubation
F i g s 5-6. Parasitism of spores of Gigaspora giganfea by Verficillium I. Fig. 5. Internal projection (IP) arising from spore wall. Bar = 20 pm. Fig. 6. Side view of IP. N o t e narrow channel (small arrow) indicating path of the parasite through the spore wall and the continuation of the channel into the IP (large arrow). Bar = 10 pm.
Table 4. Summary of pathogenicity bioassays based on condition of spores from which test microorganisms were isolated Number of species forming Conditiona
n"
IP
FRC
no invasionc
Healthy Mottled Brown Dead
4 13
4 10 5 2
0 7 4 2
0 3 4 2
9
6
Proportion of species forming IP or FRC 1
0.77 0.56 0.67
Condition of spore of Gigaspora gigantea from which the test organism was h s t isolated. Number of species tested. One species, Geomyces pannorum, is included twice. One isolate was from mottled spores and one from brown spores. ~ l l other species are represented by a single isolate. Isolates in this category are considered non-pathogenic. a
of the parasite were much reduced in diameter inside the canal when compared to their diameter on entrance or exit (Fig. 6). The development of fine radial canals (FRC) appeared not to elicit the deposition of wall material that is characteristic of the IP. During their formation, FRC arose as the hypha of a parasite narrowed in diameter and penetrated all three walls of the spore (Figs 1, 2) (Lee, 1991).
Verticillium sp. Acremonium sp. Sporothrix sp. I Actinomycete I Cladosporium sp. Chrysosporium parvum Cercosporella persicae Geomyces pannorum I Nocardia sp. Sporothrix sp. 11 Oidiodendron sp. Streptomyces sp. I Epicoccum sp. mycelia sterilia I Arfhriniutn phaeospermum Geomyces pannorum I1 Spicaria lilacinum lsaria sp. I Geotrichum candidum Streptomyces sp. I1 Actinomycete I1
3 months
4 months
47.1' (8) 35.7 (10) 10.2 (13) 8.0 (9) 3-5 (15) 3.4 (7) 3.4 (16)
n.d.** n.d. 1.4 (14) 6.7 (9) 5.1 (14) 3.3 (9) n.d.
Mean number of intemal projections (IP)per spore. Values in parentheses indicate the number of spores examined. '* n.d., not determined.
Isolation of parasitic micro-organisms from spores used in the bioassay
All 22 species of micro-organisms that parasitised healthy spores of G. giganfea in the bioassay were successfully reisolated. No other species was isolated from spores used in the bioassays. Time-course experiment
The number and length of IP increased with time (Table 7). Short IP were formed in as little as one week in response to attempted penetration by Verficillium hyphae. The mild heat treatment had no effect on susceptibility of spores to parasitism by Verticillium in the first 4-6 wk of
Parasitism of spores of Gigaspora gigantea
< 0.8 prn) after exposure to hyphae of the same two parasites. These dead spores soon (within ca 1 month) became filled with hyphae of the parasitic fungi. DISCUSSION
Figs 7-9. Parasitism of spores of Gigaspora giganfea. Fig. 7. Surface view of spore with numerous internal projections (IP) induced by Sfrepfomycessp. I. Note narrow channel in the centre of each IP and the presence of two channels in some IP (arrows). Bar = 10 vm. Fig. 8. Three internal projections induced by Sfrepfomycessp. Note concentric layers of cell wall deposition and narrow channel in IP indicated by the arrow. Bar = 10 vm. Fig. 9. IP (arrow) formed on a germ tube of G. giganfea in response to Chrysosporium parvurn. Bar = 20 pm.
Table 7. Time-course experiment: formation of intemal projections (IP) in heat-treated (HT) or untreated (NHT) healthy spores of Gigaspora gigantea in response to Verticillium sp. I Number of IP Duration of exposure (wk) 1
2 4 6 7 9
Length of IP
(m)
HT
NHT
HT
NHT
0" (30) 1.5 (30) 5.6 (16) 11.4 (16) 1 9 0 (28) 25.0 (30)
1.1 (32) 2.2 (17) 6.4 (31) 10.5 (28) 12.5 (28) 14.0 (30)
05.9 17.6 18.7 30.0 36.4
4.2 5.5 13'4 16.3 21.2 22.0
Mean of number of IP per spore. Values in parentheses indicate the number of spores examined. ** Average length (pm) of 1P.
incubation. By the seventh and ninth weeks, however, heattreated spores had significantly more and longer IP than did untreated spores ( P < 0.05). Response of live and dead spores
IP were formed only when live spores were parasitised. Following IP formation induced by Verticillium sp. or Acremonium sp., there was little hyphal proliferation within the G. gigantea spores. FRC also were formed in live spores when cultured for 3-4 months. In contrast, spores that were killed by heat never formed IP, but were riddled with FRC (diam.
A variety of common soil fungi were intimately associated with the spores of G. gigantea collected from a sand dune site. Nearly fifty species were isolated from surface-disinfected spores, leading us to suggest that the micro-organisms were living within the spores. The fact that healthy spores had fewer associated species than did older, moribund and dead spores supported this suggestion. Other investigators have surveyed the population of micro-organisms associated with the spores of AMF or occurring in soil in pot cultures of AMF (Krishna et al., 1982; Secilia & Bagyaraj, 1987, 1988; Ames, 1989; Ames, Mihar & Bayne, 1989). However, none of the isolated micro-organisms in these studies was tested for ability to parasitise healthy spores. Ames (1989) was the first t o survey the population of micro-organisms associated with washed AM fungal spores that were not obviously paras~tised.He recovered 5 1 isolates of chitin-decomposing micro-organisms from spores of Glomus macrocarpum, but these were not assessed for pathogenicity. Actinomycetes and bacteria constituted 99% of the microorganisms isolated. Spores of G. gigantea in sand dunes in Rhode Island routinely pass from the healthy state of newly formed spores over a period of ca 7 months to dead, blackened spores in four distinguishable steps: healthy, greenish yellow; yellow with brown spots (mottled); reddish-orange-brown; and dead. The sequence is not due to germination (Lee & Koske, 1994), but results from the activities of soil micro-organisms. While only fungi and actinomycetes were shown to be capable of causing symptoms of ageing and death of spores in this study, other soil organisms such as bacteria, protists and microfauna also may be important causal agents in some soils (Gerdemann & Trappe, 1974; McIlveen & Cole, 1976; Old & Wong, 1976; Wamock, Fitter & Usher, 1982; Ames, Reid & Ingham, 1984; Moore, St John, & Coleman, 1985; Rabatin & Stinner, 1985, 1988; Secilia & Bagyaraj, 1988; Ames, 1989; Arnes, Mihara & Bayne, 1989; Boyetchko & Tewari, 1991). Actinomycetes and soil fungi have long been thought to be capable of parasitizing the spores of AMF (Malencon, 1947; Petitberghein, 1956; Mosse, 1956; Godfrey, 1957), but fewer than 10 species of micro-organisms previously have been shown to be parasites of AMF spores. A high percentage (77%) of the micro-organisms isolated from spores of Gigaspora gigantea were found to be capable of functioning as parasites. Species varied in their pathogenicity, but most were able to penetrate healthy spores either through internal projections or fine radial canals. Verticillium sp. and Acremonium sp. were the most virulent species isolated. Both genera are known to include mycoparasitic species (e.g. Smith & Davidson, 1979; Van Zaayenn & Gams, 1982). These two species were the ones most frequently isolated from spores of G. gigantea collected from the field over a period of one year. The seasonal abundance of these species in the field (based on isolations from spores) generally coincided inversely with the
PauJu Lee and R. E. Koske abundance of healthy spores of G . giganfea in the field (Lee, 1991). The greater number of micro-organisms isolated from older spores probably reflects the decreased ability of aged spores to defend themselves against parasites (Lee, 1991).This would account for the great dissimilarity between species isolated from healthy and mottled spores compared with brown and dead spores. The results of the pathogenicity assays of various microorganisms confirmed earlier suggestions that IP and FRC were localized reactions or formations in the spore wall as a result of attempted penetration by parasites (Petitberghein, 1956; Godfrey, 1957; Tzean & Chu, 1985). Internal projections were first illustrated in spores of AMF by Bucholtz (1912). Since then, numerous investigators have illustrated them (e.g., Mosse, 1956; Godfrey, 1957; Mosse & Bowen, 1968; Khan, 1971; Gerdemann & Bakshi, 1976; Tzean & Chu, 1985; Koske, 1985), but they have been observed previously to form in response only to one species, the chytrid Spizellomyces acuminafus (Tzean & Chu, 1985). In this study, IP were formed only by live spores in response to attempted penetration by a variety of soil fungi and actinomycetes. Godfrey (1957) suggested that IP formed only when a spore was parasitised before the spore wall had reached its final thickness (i.e. immature spore) and thus was readily able to respond by depositing wall material at the point of attack. From the results of this study, it appears that fully formed, mature, living spores also are able to form IP. The IP formed by spores of G. giganfea bore great morphological and functional similarity to the lignitubers produced by higher plants. Mosse & Bowen (1968) previously illustrated IP in AMF spores, referring to them as 'lignituberlike ingrowths'. True lignitubers are formed in root cells of plants in response to attempted penetration by parasites. Callose and cellulose are synthesized de novo at the point of attack, eventually becoming infused with phenolic compounds. The resultant structure, the lignituber, thus may envelop and contain the penetrating hyphae (Roberts & Boothroyd, 1984; Agrios, 1988). The IP found in spores appear to form in a similar manner, but details at the biochemical level are lacking. The length of an IP in a spore can increase with age as the parasite advances and the host (spore) responds with continued depositions of material at the apex of the IP. Different parasites induced IP of different lengths, perhaps reflecting the ability of each parasite to overcome the host defences. Species that induce short IP may either be easily halted in their penetration by the host (indicating a successful defence) or may be especially aggressive, breaking through the end of the IP and entering the host cytoplasm. Long IP, such as those formed in response to Verticillium, probably indicate a h e balance between penetration and wall deposition. The success of the parasite in entering the spore cytoplasm was not assessed in comparison to IP length in this study. FRC were formed both in dead and live spores. When FRC developed in living spores, it appeared that the parasite elicited no visible response by the host. In contrast, the hyphae of species that induced IP immediately stimulated IP formation as the hypha penetrated the inner spore wall. FRC previously have been demonstrated to be induced by three
465 species (Rhizidiomycopsis sfomafosa, Spizellomyces acuminafus, and S. punctatum (Ross & Ruttencutter, 1977; Sparrow, 1977; Schenck & Nicolson, 1977; Tzean, Chu & Su, 1983). although the last species is now thought to be more saprotrophic than parasitic (Paulitz & Menge, 1984). In our study, pathogenic species were more frequently isolated from healthy or mottled spores. Species isolated only from brown or dead spores typically were not pathogenic. These species (e.g. Fusarium sp. Gliomastix sp., Morfierella ramanniana, and Trichoderrna sp.) appear to be saprobes invading already parasitised spores. An exception was Humicola fuscoafra, a parasitic species isolated only from dead spores of G. gigantea. Parasitism of spores of Glornw deserficola (as G. fasciculafum (see Trappe, Bloss & Menge (1984)) by H. fuscoafra was reported by Daniels & Menge (1980). The parasite entered the spores of this fungus through the hyphal attachment and did not form FRC or IP in the spore wall. In our study, hyphae of H. fuscoatra formed abundant FRC in the spores of G . giganfea. No entry through the hyphal attachment was observed. These results suggest that parasitism by fungi and actinomycetes may be an important component of the decline in spore abundance of G. giganfea in sand dunes during spring and summer. This work lends further evidence to support the view that parasitic micro-organisms can significantly reduce the inoculum potential of A M fungi in field soils (Ross & Ruttencutter, 1977; Bagyaraj & Menge, 1978; Daniels, 1981; Paulitz & Menge, 1984, 1986; Hetrick, et al., 1986, 1988; McGonigle & Fitter, 1988; Lee & Koske, 1994). We thank Dr Jane Gemma for many helpful suggestions and advice in the lab work and Dr Roger Goos for taxonomic assistance and encouragement.
REFERENCES Agrios, G. N. (1988). Plant Pathology. 3rd ed. Academic Press: New York. Ames, R. N. (1989). Mycorrhiza development in onion in response to inoculation with chitin-decomposing actinomycetes. New Phytologist 112, 423-427. Ames, R. N., Mihar, K. L. 81 Bayne, H. G. (1989). Chitin-decomposing actinomycetes associated with a vesicular-arbuscular mycorrhizal fungus from a calcareous soil. New Phytologist 111,67-71. Ames, R. N. Reid, C. P. P. & Ingham, E. R. (1984). Rhizosphere bacterial population responses to root colonization by a vesicular-arbuscular rnycorrhizal fungus. New Phytologist 96, 555-563. Bagyaraj, D. J. & Menge, 1. A. (1978). Interaction between a VA mycorrhiza and h t o b a c t e r and their effects on rhizosphere microflora and plant growth. New Phytologist SO, 567-573. Bandoni, R. (1972). Terrestrial occurrence of some aquatic hyphomycetes. Canadian lournal of Botany 5 0 , 2283-2288. Ban; D. J. S. (1984). The classification of Spizellomyces, Gaertneriomyces, Triparticalcar, and Kochiomyces (Spizellornycetales, Chytridiornycetes). Canadian Journal of Botany 62,1171-1201. Barr, D.J. S. (1987). Isolation, culture and identification of Chytridiales, Spizellomycetales, and Hyphochytriales. In Zoosporic Fungi in Teaching GResearch (eds. M. S. Fuller & A. Jaworski), pp. 118-204. southeastern Publishing Corporation: Athens, Georgia. Barron, G. L. (1968). The Genera of Hyphomycetes from Soil. The Williams & Wilkins Company: Baltimore, Maryland. Bo~etchko,S. M. & Tewari, J. P. (1991).Parasitism of spores of the vesiculararbuscular mycorrhizal fungus, Glomw dimorphicum. Phytoprotection 72, 27-32. M Y C 98
Parasitism of spores of Gigaspora giganfea Bray, J. R. & Curtis, J. T. (1957). An ordination of the upland forest communities of southern Wisconsin. Ecological Monographs 27,325-349. Bucholtz, P. (1912). Beitrage zur Kenntnis der Gattung Endogone Link. Beihefte rum Botanischen Centralblatt 39,147-224. Campbell, M. C. & Stewart, J. L. (1980). The Medical Mycology Handbook. J. Wiley & Sons: New York. Carmichael, J. W., Kendrick, W. B., Conners, I. L. & Sigler, L. (1980). Genera of Hyphomycetes. University of Alberta Press: Edmonton, Alberta. Daniels, B. A. (1981). The influence of hyperparasites of vesicular-arbuscular mycorrhizal fungus on growth of citrus. Phytopathology 71, 212-213. Daniels, B. A. & Menge, J. A. (1980). Hyperparasitism of vesicular-arbuscular mycorrhizal fungus. Phyt~~athology 70,584-588. Gemma, J. N., Koske, R. E. & Carreiro, M. (1989). Seasonal dynamics of selected species of V-A mycorrhizal fungi in a sand dune. Mycological Research 94,27-32. Gerdemann, J. W. & Bakshi, B. A. (1976). The Endogonaceae of India: two new species. Transactions of the British Mycological Society 66,340-343. Gerdemann, J. W. & Trappe, J. M. (1974). The Endogonaceae in the Pacific Northwest. Mycologia Memoirs 5, 1-76. Godfrey, R. M. (1957). Studies of British species of Endogone. 11. Fungal parasites. Transactions of the British Mycolog~calSociety 40, 136144. Hetrick, B. A. D., Kitt, D. G. & Wilson, G. T. (1986). The influence of phosphorus fertilization, drought, fungal species and soil microorganisms on mycorrhizal growth response in tallgrass prairie plants. Canadian Journal of Botany 64,1199-1203. Hetrick, 8. A. D., Wilson, G. T., Kitt, D. G. & Schwab, A. P. (1988). Effects of soil microorganisms on mycorrhizal distribution and growth of big bluestem grass in non-sterile soil. Soil Biology and Biochemistry 20,501-507. Khan, A. G. (1971). Occurrence of Endogone spores in West Pakistan soils. Transactions of the British Mycological Society 56, 217-224. Koch, W. J. (1972). Fungi in fhe Laboratory, a Manual and Text. University of North Carolina Student Stores: Chapel Hill, North Carolina. Koske, R. E. (1981). Gigaspora gigantea: observations on spore germination of a V-A mycorrhizal fungus. Mycologia 73,288-300. Koske, R. E. (1985). Glomw aggregatum emended: a distinct taxon in the Glomw fasciculatum complex. Mycologia 77, 619-630. Koske, R. E. (1987). Distribution of vesicular-arbuscular mycorrhizal fungi along a latitudinal temperature gradient. Mycologia 79,5 5 4 8 . Koske, R. E. & Halvorson, W. L. (1981). Ecological studies of vesiculararbuscular mycorrhizae in a barrier sand dune. Canadian Journal of Botany 59, 1413-1422. Krishna, K. R., Balakrishna, A. N. & Bagyaraj, D. J. (1982). Interaction between a vesicular-arbuscular mycorrhizal fungus and Sfreptomyces cinnarnomew and their effects on finger millet. New Phytologist 92,401-405. Lee, P. J. (1991). Seasonal dynamics of Gigaspora gigantea and associated microorganisms in a sand dune. Ph.D. Thesis. The University of Rhode Island, Kingston, RI. Lee, P. J. & Koske, R. E. (1994). Gigaspora gigantea: seasonal abundance and ageing of spores in a sand dune. Mycological Research 98,453-457. Malencon, M. C. (1947). Etudes de parasitisme mycopathologique. Revue de Mycologie (Paris) 7, 27-52. McGonigle, T. P. & Fitter, A. H. (1988). Ecological consequences of arthropod grazing on VA mycorrhizal fungi. Proceedings of the Royal Society of Edinburgh 94B,25-32. Mcllveen, W. D. & Cole, H. (1976). Spore dispersal of Endogonaceae by worms, ants, wasps and birds. Canadian Iournal of Botany 54, 1486-1489. Moore, J. C., St John, T. V. & Coleman, D. C. (1985). Ingestion of vesiculararbuscular mycorrhizal hyphae and spores by soil microarthropods. Ecology 66, 1979-1981. Mosse, B. (1956). Fructifications of an Endogone species causing endotrophic mycorrhizas in fruit plants. Annals of Botany 20,349-362. (Accepted 23 September 1993)
466 Mosse, B. & G. D. Bowen. (1968). A key to the recognition of some Endogone spore types. Transactions of the British Mycological Society 51,469-483. Old, K. M. & Wong, J. N. F. (1976).Perforation and lysis of fungal spores in natural soils. Soil Biology and Biochemistry 8,285-292. Omar, M. B., Bolland, M. B. L. & Heather, W. A. (1979). A permanent mounting medium for fungi. Bulletin of the British Mycological Society 13, 31-32. Paulitz, T. C. & Menge, J. A. (1984). Is Spizellomycespunctatum a parasite or saprophyte of vesicular-arbuscular mycorrhizal fungi? Mycologia 76, 99-107. Paulitz, T. C. & Menge, J. A. (1986). The effects of a mycoparasite on the mycorrhizal fungus Glomw deserticola. Phytopathology 76,351-354. Petitberghein, P. (1956). Les champignons hypoges de la region de Dijon. Bulletin Scient$que de Bourgogne 16, 113-155. Rabatin, S. C. & Stinner, B. R. (1985). Arthropods as consumers of vesiculararbuscular mycorrhizal fungi. Mycologia 77,320-322. Rabatin, S. C. & Stinner, B. R. (1988). Indirect effects of interactions between VAM fungi and soil-inhabiting invertebrates on plant processes. Agriculture, Ecosystems and Environment 24, 135-146. Roberts, D. A. & Boothroyd, C. W. (1984). Fundamentals of Plant Pathology. 2nd ed. W. H. Freeman and Company: New York. Ross, J. P. & Daniels, B. A. (1982). Hyperparasitism of endomycorrhizal fungi. In Methods and Principles of Mycorrhizal Research (ed. N. C. Schenck), pp. 55-58. American Phytopathological Society: St Paul, Minnesota. Ross, J. P. & Ruttencutter, R. (1977). Population dynamics of two vesiculararbuscular mycorrhizal fungi and the role of hyperparasitic fungi. Phytopathology 67,490-496. Schenck, N.C. & Nicolson, T. H. (1977). A zoosporic fungus occurring on species of Gigaspora margarita and other vesicular-arbuscular mycorrhizal fungi. Mycologia 69, 1049-1053. Secilia, J. & Bagyaraj, D. J. (1987). Bacteria and Actinomycetes associated with pot cultures of vesicular-arbuscular mycorrhizas. Canadian lournal of Microbiology 33, 1069-1073. Secilia, J. & Bagyaraj, D. J. (1988). Fungi associated with pot cultures of vesicular-arbuscular mycorrhizas. Transactions of the British Mycological Sociefy 90,117-119. Siqueira, J. 0.. Hubbell, D. H., Kimbrough, J. W. & Schenck, N. C. (1984). Stachybohys chartarum antagonistic to azygospores of Gigaspora margarita in vitro. Soil Biology and Biochemistry 16,679-581. Smith, J. D. & Davidson, G. N. (1979). Acremonium boreale n. sp., a sclerotial low-temperature-tolerant, snow mold antagonist. Canadian ]oumal of Botany 57,2122-2139. Southwood, T. R. E. (1978). Ecological Methods, with Particular Reference to The Study of Insect Populations. 2nd ed. J. Wiley & Sons: New York. Sparrow, F. K. (1977). A Rhizidiomycopsis on azygospores of Gigaspora margarita. Mycologia 69,1053-1958. Trappe, J. M., Bloss. H. E. & Menge, I. A. (1984). Glomw deserticola sp. nov. Mycotaxon 20, 123-127. Tzean, S. S. & Chu, C. L. (1985). Ultrastructure of parasitism of vesiculararbuscular mycorrhizai fungi by Phlyctochytriumplurigibbosum. In Proceedings of the 6th North American Conference on Mycorrhizae (ed. R. Molina), p. 443. Forest Research Laboratory, Oregon State University: Cowallis, Oregon. Tzean, S. S., Chu, C. L. & Su, H. J. (1983). Siroplasmalike [sic]organisms in a vesicular-arbuscular fungus and its mycoparasite. Phytopathology 73, 989-991. Van Zaayen A. & Gams, W. (1982). Contribution to the taxonomy and pathogenicity of fungicolous Verticillium species. 11. Pathogenicity. Netherlands Journal of Plant Pathology 88, 143-154. Wamock, A. J., Fitter, A. H.& Usher, M. B. (1982). The influence of a springtail Folsomia candida (Insecta, Collembola) on the mycorrhizal association of leek Allium pomm and the vesicular-arbuscular mycorrhizal endophyte Glomus fasciculatw. New Phytologist 90,285-292.
Mycol. Res. 98 (4): 458-466 (1994) Printed in Great Britain
Gigaspova gigantea: parasitism of spores by fungi and actinomycetes
PAU-JU LEE' A N D R.E. KOSKEY Department of Botany, University of Rhode Island, Kingston, Rhode Island 02881, U.S.A.
Fungi and actinomycetes were isolated monthly for one year from spores of the arbuscular mycorrhizal fungus Gigaspora gigantea recovered from a maritime sand dune. Spores in four different stages of vigour (newly formed, greenish-yellow healthy; yellow, moribund, mottled with brown spots; brown; and dead, blackened and collapsed) were used for isolation. From 272 isolates cultured from crushed, surface-disinfected G. gigantea spores, 44 species of fungi and six actinomycete species were recovered. The five most frequently isolated organisms were Acremoniurn sp., Chrysosporium paruum, Exophiala werneckii, Trichoderma sp. and Verticillium sp. The species lists derived from the four spore types differed significantly. Thirty-one of the isolated species were tested For their ability to parasitise healthy G. gigantea spores and to invade spores killed by a hot water treatment. Twenty-two species could function as pathogens, forming intemal projections (IP), fine radial canals (FRC) or both in the spore wall. IP were induced by Acremoniurn sp., Chysosporium parvum, Cladosporium sp., Geomyces pannorum, Oidiodendron sp., Sporothrix sp., Verticillium sp. and by the actinomycetes Nocardia sp. and Streptomyces sp. The IP length was positively correlated with duration of exposure to the parasites. In the bioassay, both IP and FRC were formed by live spores, while heat-killed spores possessed only FRC after penetration by the test microorganisms. Pathogenicity differed among the parasites and was greatest for Verticillium and Acremoniurn.
Observations suggest that parasitic fungi and actinomycetes may significantly reduce populations of spores of arbuscular mycorrhizal fungi (AMF) in the field and induce adverse effects on mycorrhizally dependent plants (Ross & Ruttencutter, 1977; Daniels & Menge, 1980; Ross & Daniels, 1982; Paulitz & Menge, 1986). However, despite the potentially great importance of such parasites, only five species of microorganisms (all of them fungi) have been shown to be capable of parasitizing spores of AMF. These include Anguillospora pseudolongissima Ranzoni (Daniels & Menge, 1980; Paulitz & Menge, 1986), Hurnicola fuscoafra Traaen (Daniels & Menge, 1980), Sfachybotrys chartarum (Ehrenb. ex Link) S. Hughes (Siqueira ef al., 1984), and two chytridiaceous species R h i z i d i o m y ~ o ~ s isfomafosa s Sparrow (Schenck & Nicolson, 1977; Sparrow, 1977) and Spizellomyces acuminafus (D. J.S. Barr) D. J. S. Barr (Barr, 1984). Two other species, Phlycfochyfrium kneipii Gaertn. (Tzean & Chu, 1985) and P. plurigibbosurn D. J. S. Barr (Tzean, Chu & Su, 1983), were reported as pathogenic to spores of AMF, but these claims appear to be based on misidentifications of S. acuminafus (Barr, pers. communication). A few other species of fungi and actinomycetes have been isolated from obviously parasitised spores of AMF and are suspected as parasites, but their ability to parasitise healthy
Current address: Department of Mathematics and Science Education, Tainan Teachers College, Tainan, Taiwan, R.O.C. * Corresponding author.
spores has not been proven experimentally. These include S. puncfafum (Koch) D. J . S. Barr ( = Phlycfochytrium puncfafum
Koch) (Ross & Ruttencutter, 1977; Daniels, 1981; Paulitz & Menge, 1984), an unidentified 'Pyfhium-like' species (Ross & Ruttencutter, 1977), a species of Cephalosporiurn (Malencon, 1947), and various unidentified fungi and actinomycetes (Mosse, 1956; Petitberghein, 1956; Godfrey, 1957). All of the parasites or suspected parasites listed above were isolated from obviously parasitised AMF spores, often from spores recovered from greenhouse-grown pot-cultures rather than from natural conditions. There has been no systematic isolation from healthy, aged, moribund, and dead spores of any AMF using material collected from the field. Species of potentially important parasitic micro-organisms may have been overlooked because of emphasis on a single category (parasitised) of spores. Two distinct morphological features of parasitised spores and hyphae of AMF have been described and thought to be induced during the penetration of the fungal wall. In one, it is presumed that the parasites create narrow channels (fine radial canals (FRC) [Petitberghein, 1956; Godfrey, 19571) in the wall of the AMF. In the second type, conical or digitate structures (internal projections (IP) [Mosse, 1956; Petitberghein, 1956; Godfrey, 1957; Mosse & Bowen, 19681) form on the inside of the spore walls where penetration occurs. These features have been observed in spores of AMF for over 80 years (Bucholtz, 1912), and the correlation between parasitism and FRC and IP was made by Mosse (1956),
Pau-Ju Lee and R. E. Koske Petitberghein (1956) and Godfrey (1957). Just three species (Rhizidiomycopsis stomafosa, Spizellomyces punctatum, and S. acuminatus [as P. kneipii]) have been shown to be capable of inducing FRC (Ross & Ruttencutter, 1977; Schenck & Nicolson, 1977; Sparrow, 1977; Tzean, Chu & Su, 1983), and only S. acuminatu (as P. plurigibbosurn) has been experimentally shown to induce IP (Tzean & Chu, 1985). The purposes of the present study were to determine which fungi and actinomycetes were intimately associated in the field with spores of Gigaspora gigantea (T. H.Nico1 & Gerd.) Gerd. & Trappe and to screen some of those isolates for pathogenicity to AMF spores. Gigaspora giganfea was selected for study because it is one of the dominant AMF species in coastal dunes of the U.S. Atlantic seaboard (e.g. Koske & Halvorson, 1981; Koske, 1987; Gemma, Koske & Carreiro, 1989). A further aim was to assess the relationship between parasitism and the presence of FRC and IP in spores.
MATERIALS A N D M E T H O D S Monthly isolation of micro-organisms from four stages of spores
Fungi and actinomycetes were isolated from spores of G. giganfea in four different stages of health from monthly collections (Mar. 1989 to Feb. 1990) from the barrier sand dune at Moonstone Beach, Rhode Island (RI). The collection area had a dense cover of American beachgrass (Ammophila breviligulafa Fern.). A stratified random sampling technique was used to collect 15 samples each month. Spores were sampled extracted and classified mostly by colour into healthy, moribund and dead spores (Lee & Koske, 1994). Moribund spores were further differentiated into mottled and completely brown categories, resulting in four life stages healthy, mottled, brown and dead. Each month 20 spores of each stage were surface-disinfected in 2 % Clorox@(0.105 % NaOCl,) (Koske, 1981) for 2-3 min followed by three rinses in sterile deionized water. To ensure that micro-organisms that were potentially pathogenic to spores of G. giganfea were isolated, only surface-disinfected spores were used. The 20 surface-disinfected spores of each stage were crushed in 1ml of half-strength MYP solution (Difco Malt Extract, 7.0 g ; Difco Soytone, 1.0 g ; Difco Yeast Extract, 0.5 g ; distilled water, 1 1) (Bandoni, 1972) to which 50 mg 1-' tetracycline (MYPT) were added to suppress bacterial growth. A 0.2 ml portion of the crushed-spore suspension in MYPT was spread evenly in each of five 100 x 15 mm Petri dishes containing 20 ml of MYPT agar (MYPT 1.5 % Difco Bacto agar). Plates were incubated for up to two weeks at room temperature (21-27 OC) and examined daily for developing colonies. Results were recorded as the number of colonies per plate. From each colony a culture was made for identification and subculturing. Identifications were made on MYPT agar by the agar slide method (Koch, 1972). Actinomycetes and hyphomycetes were identified only to genus (Barron, 1968; Campbell & Stewart, 1980; Carmichael ef al., 1980). Permanent slides were prepared by mounting isolated micro-organisms in a polyvinyl alcohol-lactic acid-glycerol solution (PVLG)
+
459 (Omar, Bolland & Heather, 1979) containing 0.5 % cotton blue. Voucher specimens are lodged in the collection of the first author. Additional healthy spores were isolated monthly from the dune and used for pathogenesis assays (see below). Attempts were made to isolate chytrids by placing brown spores in Petri dishes containing autoclaved tap water or pond water baited with pine pollen or other natural substrates such as snake skin, grass straw, or surface-disinfested spores of G. giganfea. Plates were incubated in a refrigerator (4') (Barr, 1987).
Data analysis
A modified Sorensons' Coefficient of Similarity (Bray & Curtis, 1957; Southwood, 1978) was calculated to compare the populations of micro-organisms isolated from spores in different stages of health. The calculations considered both presence and abundance of each species. Abundance was determined by measuring the number of colonies of each species that developed on the isolation plates. Similarity was calculated by the formula:
where aN = the total number of colonies developing on the 5 Petri dishes plated with spore suspension from spores in stage 'a' (i.e. healthy, mottled, brown, or dead), and bN is the total number of colonies from spores in stage 'b' and cN is the number of colonies in common.
Pathogenicity testing of isolated micro-organisms
In this study, the term pathogenicity is defined as the successful establishment of a micro-organism thallus on or in the living spore of G. giganfea as shown by the formation of IP, FRC, or both in the spore wall. Micro-organisms able to colonize live spores were thus regarded as pathogens. Of the 50 species of micro-organisms isolated from spores of G. gigantea, 31 species (including two isolates of one species, Geomyces pannorum) were selected for pathogenicity testing (Table 1).Micro-organisms were initially sorted into three groups on the basis of hyphal diameter: < 1 pm, 1-2 pm, and > 2 pm. Preliminary observations of the diameter of the FRC and the canals of IP of parasitised spores, indicated that most pathogenic isolates would fall into the first two categories. Greenish-yellow, healthy spores of G. gigantea, previously surface-disinfected with 0.105 % NaOCl,, were exposed to active cultures of the test organisms to assess their pathogenicity. The spores were freshly isolated from a sand dune soil (Moonstone Beach, RI) by wet-sieving and were exposed to a brief heat treatment before use in the pathogenicity test. In preliminary tests, untreated spores were slow to become parasitised, although later tests showed otherwise, and a heat treatment (immersion in a water bath at 35' for 5 min) which caused little loss in viability (Lee, 1991) was performed to make them more susceptible to parasitism. For the pathogenicity assay, a 100 x 20 mm Petri dish containing 20 ml of water agar (Difco Bacto agar, 15 g ;
Parasitism of spores of Gigaspora gigantea Table 1. Micro-organisms isolated from spores of Gigaspora gigantea
Stagesb
NO.^
*mycelia sterilia I 'mycelia sterilia I1
B, D M, 8, D
4
Actinomycetes 'Nocardia sp. 'Streptomyces sp. I *Streptornyces sp. I1 'Sfreptomyces sp. I11 'unidentified sp. I 'unidentified sp. 11
M M, 8, D M M H, M, B, D B
1 5 1 1
Av. colsd
Fungi 'Acremonium sp. *Arthrinium phaeospermum Aspergillus flawus Aspergillus nidulans Aspergillus terrew Aspergillus versicolor *Cercosporella persicae Chaetomium sp. 'Chysosporium parvum 'Chdosporium sp. Cylindrocarpon sp. "Epicoccum sp. *Exophiah werneckii Fonseceae pedrosoi 'Fusarium sp. I Fusarium sp. I1 Fusicladium sp. Fusidium sp. *Geomyces pannorum I "Geomyces pannorum I1 'Geotrichum candidum 'Gliomastix murorum "Gliomastix sp. 'Humicola fuscoatra Hyalodendron sp. "Isaria sp. I Isaria sp. I1 Monacrosporium sp. Monilia sp. 'Mortierella ramanniana 'Oidiodendron sp. Papulospora sepedonioides Penicillium sp. 'Phialophora sp. Piedraia hortae Septonema sp. *Spicaria lilacinum 'Sporothrix sp. 'Sporothrix sp. I1 Stemphylium sp. 'Trichoderma sp. "Tripospermum sp. *Verticillium sp. I Verticillium sp. I1 7.3 f16.1 2.8 f8.3
2
0-lf0.3 0.6f 0.8 0.1k0.3 0.1 k 0.3 0.4f0.7 0.1 f0.3
4
1
* indicates species or isolate used
in pathogenicity tests. Stage of spores of Gigaspora gigantea from which the listed microorganisms were recovered. H, healthy; M, mottled; B, brown; D, dead. Number out of 12 months sampled in which the microorganism was isolated. Average number of colonies isolated per month s . ~ .
+
deionized water, 1 litre) was inoculated with a 6 rnrn diameter disk cut from the margin of a 7-day-old colony of the test organism growth on MYP agar. Ten to 20 surface-disinfected, yellow (therefore assumed not parasitised) spores of G. gigantea were placed on the water agar surface 5-10 mm from the edge of the inoculum disk. As controls, spores were arranged around a disk of sterile MYP agar. Each treatment was replicated three or more times. Plates were incubated at room temperature (21-27O) for three months, when spores were recovered and mounted in PVLG with or without 0.05 % cotton blue and examined microscopically. Parasitic potential of micro-organisms was assessed by counting the number of FRC or the number and length (vm) of IP that occurred on each spore. The 11 most pathogenic species in the above experiments were re-tested using the same bioassay, each using 10-20 spores replicated three times. Petri plates were sampled after 0.5 to 3 months, depending on the rapidity of mottling and browning, and the percentage of spores with IP or FRC was determined. Preliminary pathogenicity assays on water agar were slow, so a similar experiment was conducted (after Daniels & Menge, 1980) with a few species of micro-organisms in autoclaved dune soil. Dune sand from Moonstone Beach was passed through a 1.5 mm sieve, washed in tap water, air dried, and autoclaved for 1h at lZ1° on two successive days. Fifteen to 20 heat-treated, surface-disinfested yellow spores were placed on a sterile filter paper (5.5 cm diam.). The filter paper was then folded in half and set in a 80 x 100 mm culture dish containing ca 250 ml of the twice-autoclaved sand (moisture content ca 20%). Five agar plugs (8-10 mm diam.) from the margin of actively growing colonies of Streptomyces sp., Acremonium sp., Chysosporium sp., and Verticilliurn sp. on MYP agar were placed on the filter paper and covered with moist, sterile sand to a depth of 10 mm. The dishes were covered, and the culture incubated at room temperature (21-27'). Controls were identical except that agar plugs were not used. Each treatment was replicated three times. Spores were removed, stained, mounted, and examined intermittently as in the previous experiment to determine parasitic infection.
Re-isolation of pathogenic micro-organisms from spores used in the bioassay To determine if the test micro-organisms had penetrated the healthy spores of G. gigantea in the pathogenicity bioassays, one or two spores from each treatment were removed from the assay plate after 3 to 4 months, surface-disinfected, and placed on MYP agar. The identity of the micro-organisms that grew from each parasitised spore was confirmed.
Time-course and heat-treatment experiment
An isolate of Verticillium previously shown to be a vigorous pathogen of spores of G. giganfea was used to determine the rate at which spores were parasitised. Assay plates of water agar were set up as previously described using heat-treated and non heat-treated spores. Spores were sampled after incubation for 1, 2, 4, 6, 7 and 9 wk. The number and length
Pau-Ju Lee and R. E. Koske of IP formed in the spores was assessed. Sixteen to 30 spores were examined for each treatment and sampling time. The variation in number of spores assessed resulted from the browning of some spores in the assay plates following surface-disinfestation.These spores appeared healthy (yellow) after exposure to the sterilizing treatment, but later (after the assay plates had been set up) turned brown. Such spores were excluded from assessment. Data were analysed for significant differences in pathogenicity between micro-organisms using one way analysis of variance (ANOVA). Response of live and dead spores
From the morphology of the IP in spores of G. giganfea it can be suggested that the structure of the IP is built up by the fungus responding to attempted invasion by a parasite. To test whether a living spore was required to form IP, both live and dead spores were placed in agar assay plates with two known IP-inducing parasites, Verticillium sp. and Acremonium sp. Spores for testing were killed by placing them in boiling water for 5-10 min until they turned brown. Boiled spores did not germinate. Spores were incubated with the test fungi for up to 4 months, stained with cotton blue or trypan blue in PVLG, and examined.
RESULTS Monthly isolation of micro-organisms
Initially, 272 isolates were cultured from the four stages of spores collected throughout the year. The 272 isolates represented fifty species of hyphomycetes and actinomycetes (Table I).The most frequently isolated species were Exophiala werneckii, Acremonium sp., Verficillium sp. I, Trichoderma sp. and Chysosporium parvum. The four most frequently isolated species also were the species that formed the most colonies in the dilution-isolation plates. The number of species associated with spores of G. gigantea increased with the age of the spore. Healthy spores had the fewest associated species (8) and dead spores the most (31). Seventeen species were isolated from mottled spores, and 24 species from brown spores. Significant differences occurred among the microfloras derived from the four different stages of spores (Table 2). Greatest similarity was found between the associated microfloras of healthy and mottled spores (S = 0-58). Brown and dead spores also showed a comparable similarity coefficient (S = 0.52). Least similarity occurred between the inhabitants of healthy and dead spores (S = 0.0007). The species isolated from dead spores differed most from those obtained from other stages. Only three species were isolated exclusively from healthy spores (Aspergillusflavus,A. versicolor, and Isaria sp. I [Table I). Ten of the 31 species isolated from dead spores were not isolated from other spore stages
Pathogenicity testing of isolated micro-organisms
There were no differences between results obtained with assays on water agar or in soil, and only results from the water agar tests are presented here. Of 32 isolates tested ( = 31 Table 2. Similarity between fungal popidations isolated from surfacedisinfected spores of Gigaspora gigantea in four stages of health using modified Simpson's Coefficient of Similarity'
Stage Stage
Healthy
Mottled Brown Dead
< 0.01
0.58
Mottled
Brown
-
-
0.11 0.08
< 0.01
0.52
a Calculation is based on presence and abundance of micro-organisms derived from spores. See text.
Table 3. Pathogenicity of micro-organisms isolated from spores of Gigaspora gigantea
EffectC
Fungi Acremonium sp. Arthrinium phaeospemum Cercosporella persicae Chysosporium parvum Chdosporium sp. Epicoccum sp. Erophiala werneckii Fusarium sp. I Geomyces pannorum I Geomyces pannorum I1 Geotrichum candidum Gliomastix murorum Gliomastix sp. Humicola fuscoatra Isaria sp. I Mortierella ramanniana Oidiodendron sp. Phialophora sp. Spicaria lilacinum Sporothrix sp. Sporothrix sp. I1 Trichoderma sp. Tripospemum sp. Verticillium sp. I
mycelia sterilia I mycelia sterilia I1 Adinomycetes Nocardia sp. Streptomyces sp. I Streptomyces sp. I1 Streptomyces sp. 111 unidentified sp. I unidentified sp. I1
Xu
Y"
IP
FRC
IH
1-2
M D M M B
+ + + + +
+
+ + +
>2 >2 1-2 > 2 > >2 >2 1-2 1-2 >2 1-2 1-2 >2 1-2 >2 1-2 1-2 >2 1-2 1-2 >2 >2 1-2
< <
-
-
+ + +
-
-
+ +
+ -
-
-
D
-
-
-
D H B H M H M M B M B
-
-
-
-
-
-
-
2
D
B B B
H D
+ + + + + -
-
+
+ +
B M l l
M M
<1
M
<1 <1 <1
M
B B
+
+
M
>2 1-2
-
+ +
+
+
-
+
+
-
+ + -
+ +
+
+
+
-
-
+ +
+
+
-
+
-
+
+
+ +
-
-
-
-
-
-
-
Diam. (w)of hyphae on MYP. Stage of spores of Gigaspora gigantea from which the listed microorganisms were recovered. H, health; M, mottled; B, brown; D, dead. Formation of internal projections (IP), fine radial canal (FRC) and internal hyphae (IH) in healthy, surface-disinfested spores of Gigaspora gigantea exposed in agar culture (see text) to the test organisms for 3 months. ' ' indicates presence, ' - ' indicates absence. a
Isolation of chyfridiaceous fungi
Attempts to isolate chytridiaceous fungi failed. Zoosporangia were not produced on any autoclaved natural material or on surface-sterilized spores.
+
Parasitism of spores of Gigaspom gigantea
F i g s 1-2. Parasitism of spores of Gigaspora gigantea by Streptomyces sp. I. Fig. 1. Fine radial canals (FRC) in face view are visible (small arrows) as are internal projections (IP) (large arrows). Bar = 50 wm. Fig. 2. Side view of fine radial canals (arrow)in wall. Interior of spore is to the right of the wall. Bar = 20 urn.
species), 23 (72%) were capable of functioning as pathogens of spores of G. giganfea (Table 3). Eleven isolates, including such common soil species or genera as Acremonium sp. Ckysosporium parvum, Cladosporium sp., Geomyces pannorum, Sporofhrix sp., Sfrepfomyces sp. I, and Verticillium sp. induced both IP and FRC (Figs 1-8). Species forming only FRC included Exopkiala werneckii and Humicola fuscoafra. Internal projections only (no FRC) were induced by ten species including Epicoccum, Geotrichum candidum, Isaria, Oidiodendron, and four species of actinomycetes. No IP, FRC or internal hyphae were formed in the spores in the controls or in spores exposed to Fusarium sp., Gliomastix sp., Mortierella mmanniana, Pkialophora sp., Sfreptomyces sp. 111, Trickoderma sp., Tripospermum sp., and two actinomycetes. Hyphal diameter on agar did not differ significantly between parasitic and non-parasitic isolates or species, although the average diameter of hyphae of parasitic isolates was slightly narrower (av. 1.64 Vm v. 1.80 ym). Of the 31 species tested for pathogenicity, those originally isolated from healthy or mottled spores more frequently were pathogenic than were species isolated from brown or dead spores (Table 4). Eighty-two per cent of species that were isolated from healthy or mottled spores were pathogenic in the bioassay (Table 4), while only 60% of the isolates from brown and dead spores were pathogenic. Species capable of inducing IP were isolated from healthy, mottled, brown and dead spores. Eleven species were assessed for their ability to parasitise healthy spores of G . giganfea over a period of two weeks to three months (Table 5). After 2 wk, 53-85% of the spores exposed to Acremonium and Verticillium were parasitised. B y three months, eight of the 11 species had parasitised 50% or more of the spores. Species were ranked by the number of IP that they induced
Figs 3-4. Parasitism of spores of Gigaspora gigantea by Verticillium sp. I. Internal projections (IP) formed in response to attempted penetration of the spore wall by the parasite. Fig. 3. Numerous
worm-like IP (Black arrow on white background) are present. Small, wart-like structures on the inner surface of the spore wall are not the result of parasitism. Bulbous base is indicated (black arrow). Bar = 30 wm. Fig. 4. Long IP typically induced by Verticillium. Bar = 10 urn.
after a 3- or 4-month incubation period (Table 6). As in the parasitism study, Verticillium and Acremonium were the most aggressive pathogens. Verticillium induced an average of 47.1 IP/spore after three months, and some spores had more than 100 IP. The average size of the IP induced by Verticillium was 4 x 120 Vm. Germ-tubes of G . giganfea also were attacked by hyphae of Verticillium (Fig. 9), and IP were formed (Fig. 9). In spores in which IP were formed, the first visible sign of parasitism was the occurrence of brown spots on the spore wall, resulting in a mottled appearance to the spore. IP developed first from the inner, germinal wall of the spores as the host deposited layers of cell wall-like material in front of the advancing penetration hypha. The material that was deposited appeared similar in composition to that of the laminated spore wall. The sequential deposition of materials at the point of attack was apparent as concentric layers that were especially evident at the tip of the IP (Fig. 8). Each IP enclosed one or two fine channels ( < 1 ym diam.) in which the parasite occurred (Figs. 3-8). Canals were branched or unbranched, and some IP included more than one canal (Fig, 7). The hyphae
Pau-Ju Lee and R. E. Koske
463 Table 5. Percentage of spores of Gigaspora gigantea parasitised by selected micro-organisms on water agar Culture time (months)
Acremonium sp. Verticillium sp. I Chysosporium parvum Actinomycete I Sporothrix sp. I Streptomyces sp. Nocardia sp. Geomyces pannorum I Streptomyces sp. I1 Cladosporium sp. Sporothrix sp. I1
n.d. n.d.
n.d. n.d.
n.d. n.d.
40 41
* n.d., not determined.
Table 6. Formation of internal projections {IP) by healthy spores of Gigaspora gigantea in response to selected micro-organisms after 3 or 4 month's incubation
F i g s 5-6. Parasitism of spores of Gigaspora giganfea by Verficillium I. Fig. 5. Internal projection (IP) arising from spore wall. Bar = 20 pm. Fig. 6. Side view of IP. N o t e narrow channel (small arrow) indicating path of the parasite through the spore wall and the continuation of the channel into the IP (large arrow). Bar = 10 pm.
Table 4. Summary of pathogenicity bioassays based on condition of spores from which test microorganisms were isolated Number of species forming Conditiona
n"
IP
FRC
no invasionc
Healthy Mottled Brown Dead
4 13
4 10 5 2
0 7 4 2
0 3 4 2
9
6
Proportion of species forming IP or FRC 1
0.77 0.56 0.67
Condition of spore of Gigaspora gigantea from which the test organism was h s t isolated. Number of species tested. One species, Geomyces pannorum, is included twice. One isolate was from mottled spores and one from brown spores. ~ l l other species are represented by a single isolate. Isolates in this category are considered non-pathogenic. a
of the parasite were much reduced in diameter inside the canal when compared to their diameter on entrance or exit (Fig. 6). The development of fine radial canals (FRC) appeared not to elicit the deposition of wall material that is characteristic of the IP. During their formation, FRC arose as the hypha of a parasite narrowed in diameter and penetrated all three walls of the spore (Figs 1, 2) (Lee, 1991).
Verticillium sp. Acremonium sp. Sporothrix sp. I Actinomycete I Cladosporium sp. Chrysosporium parvum Cercosporella persicae Geomyces pannorum I Nocardia sp. Sporothrix sp. 11 Oidiodendron sp. Streptomyces sp. I Epicoccum sp. mycelia sterilia I Arfhriniutn phaeospermum Geomyces pannorum I1 Spicaria lilacinum lsaria sp. I Geotrichum candidum Streptomyces sp. I1 Actinomycete I1
3 months
4 months
47.1' (8) 35.7 (10) 10.2 (13) 8.0 (9) 3-5 (15) 3.4 (7) 3.4 (16)
n.d.** n.d. 1.4 (14) 6.7 (9) 5.1 (14) 3.3 (9) n.d.
Mean number of intemal projections (IP)per spore. Values in parentheses indicate the number of spores examined. '* n.d., not determined.
Isolation of parasitic micro-organisms from spores used in the bioassay
All 22 species of micro-organisms that parasitised healthy spores of G. giganfea in the bioassay were successfully reisolated. No other species was isolated from spores used in the bioassays. Time-course experiment
The number and length of IP increased with time (Table 7). Short IP were formed in as little as one week in response to attempted penetration by Verficillium hyphae. The mild heat treatment had no effect on susceptibility of spores to parasitism by Verticillium in the first 4-6 wk of
Parasitism of spores of Gigaspora gigantea
< 0.8 prn) after exposure to hyphae of the same two parasites. These dead spores soon (within ca 1 month) became filled with hyphae of the parasitic fungi. DISCUSSION
Figs 7-9. Parasitism of spores of Gigaspora giganfea. Fig. 7. Surface view of spore with numerous internal projections (IP) induced by Sfrepfomycessp. I. Note narrow channel in the centre of each IP and the presence of two channels in some IP (arrows). Bar = 10 vm. Fig. 8. Three internal projections induced by Sfrepfomycessp. Note concentric layers of cell wall deposition and narrow channel in IP indicated by the arrow. Bar = 10 vm. Fig. 9. IP (arrow) formed on a germ tube of G. giganfea in response to Chrysosporium parvurn. Bar = 20 pm.
Table 7. Time-course experiment: formation of intemal projections (IP) in heat-treated (HT) or untreated (NHT) healthy spores of Gigaspora gigantea in response to Verticillium sp. I Number of IP Duration of exposure (wk) 1
2 4 6 7 9
Length of IP
(m)
HT
NHT
HT
NHT
0" (30) 1.5 (30) 5.6 (16) 11.4 (16) 1 9 0 (28) 25.0 (30)
1.1 (32) 2.2 (17) 6.4 (31) 10.5 (28) 12.5 (28) 14.0 (30)
05.9 17.6 18.7 30.0 36.4
4.2 5.5 13'4 16.3 21.2 22.0
Mean of number of IP per spore. Values in parentheses indicate the number of spores examined. ** Average length (pm) of 1P.
incubation. By the seventh and ninth weeks, however, heattreated spores had significantly more and longer IP than did untreated spores ( P < 0.05). Response of live and dead spores
IP were formed only when live spores were parasitised. Following IP formation induced by Verticillium sp. or Acremonium sp., there was little hyphal proliferation within the G. gigantea spores. FRC also were formed in live spores when cultured for 3-4 months. In contrast, spores that were killed by heat never formed IP, but were riddled with FRC (diam.
A variety of common soil fungi were intimately associated with the spores of G. gigantea collected from a sand dune site. Nearly fifty species were isolated from surface-disinfected spores, leading us to suggest that the micro-organisms were living within the spores. The fact that healthy spores had fewer associated species than did older, moribund and dead spores supported this suggestion. Other investigators have surveyed the population of micro-organisms associated with the spores of AMF or occurring in soil in pot cultures of AMF (Krishna et al., 1982; Secilia & Bagyaraj, 1987, 1988; Ames, 1989; Ames, Mihar & Bayne, 1989). However, none of the isolated micro-organisms in these studies was tested for ability to parasitise healthy spores. Ames (1989) was the first t o survey the population of micro-organisms associated with washed AM fungal spores that were not obviously paras~tised.He recovered 5 1 isolates of chitin-decomposing micro-organisms from spores of Glomus macrocarpum, but these were not assessed for pathogenicity. Actinomycetes and bacteria constituted 99% of the microorganisms isolated. Spores of G. gigantea in sand dunes in Rhode Island routinely pass from the healthy state of newly formed spores over a period of ca 7 months to dead, blackened spores in four distinguishable steps: healthy, greenish yellow; yellow with brown spots (mottled); reddish-orange-brown; and dead. The sequence is not due to germination (Lee & Koske, 1994), but results from the activities of soil micro-organisms. While only fungi and actinomycetes were shown to be capable of causing symptoms of ageing and death of spores in this study, other soil organisms such as bacteria, protists and microfauna also may be important causal agents in some soils (Gerdemann & Trappe, 1974; McIlveen & Cole, 1976; Old & Wong, 1976; Wamock, Fitter & Usher, 1982; Ames, Reid & Ingham, 1984; Moore, St John, & Coleman, 1985; Rabatin & Stinner, 1985, 1988; Secilia & Bagyaraj, 1988; Ames, 1989; Arnes, Mihara & Bayne, 1989; Boyetchko & Tewari, 1991). Actinomycetes and soil fungi have long been thought to be capable of parasitizing the spores of AMF (Malencon, 1947; Petitberghein, 1956; Mosse, 1956; Godfrey, 1957), but fewer than 10 species of micro-organisms previously have been shown to be parasites of AMF spores. A high percentage (77%) of the micro-organisms isolated from spores of Gigaspora gigantea were found to be capable of functioning as parasites. Species varied in their pathogenicity, but most were able to penetrate healthy spores either through internal projections or fine radial canals. Verticillium sp. and Acremonium sp. were the most virulent species isolated. Both genera are known to include mycoparasitic species (e.g. Smith & Davidson, 1979; Van Zaayenn & Gams, 1982). These two species were the ones most frequently isolated from spores of G. gigantea collected from the field over a period of one year. The seasonal abundance of these species in the field (based on isolations from spores) generally coincided inversely with the
PauJu Lee and R. E. Koske abundance of healthy spores of G . giganfea in the field (Lee, 1991). The greater number of micro-organisms isolated from older spores probably reflects the decreased ability of aged spores to defend themselves against parasites (Lee, 1991).This would account for the great dissimilarity between species isolated from healthy and mottled spores compared with brown and dead spores. The results of the pathogenicity assays of various microorganisms confirmed earlier suggestions that IP and FRC were localized reactions or formations in the spore wall as a result of attempted penetration by parasites (Petitberghein, 1956; Godfrey, 1957; Tzean & Chu, 1985). Internal projections were first illustrated in spores of AMF by Bucholtz (1912). Since then, numerous investigators have illustrated them (e.g., Mosse, 1956; Godfrey, 1957; Mosse & Bowen, 1968; Khan, 1971; Gerdemann & Bakshi, 1976; Tzean & Chu, 1985; Koske, 1985), but they have been observed previously to form in response only to one species, the chytrid Spizellomyces acuminafus (Tzean & Chu, 1985). In this study, IP were formed only by live spores in response to attempted penetration by a variety of soil fungi and actinomycetes. Godfrey (1957) suggested that IP formed only when a spore was parasitised before the spore wall had reached its final thickness (i.e. immature spore) and thus was readily able to respond by depositing wall material at the point of attack. From the results of this study, it appears that fully formed, mature, living spores also are able to form IP. The IP formed by spores of G. giganfea bore great morphological and functional similarity to the lignitubers produced by higher plants. Mosse & Bowen (1968) previously illustrated IP in AMF spores, referring to them as 'lignituberlike ingrowths'. True lignitubers are formed in root cells of plants in response to attempted penetration by parasites. Callose and cellulose are synthesized de novo at the point of attack, eventually becoming infused with phenolic compounds. The resultant structure, the lignituber, thus may envelop and contain the penetrating hyphae (Roberts & Boothroyd, 1984; Agrios, 1988). The IP found in spores appear to form in a similar manner, but details at the biochemical level are lacking. The length of an IP in a spore can increase with age as the parasite advances and the host (spore) responds with continued depositions of material at the apex of the IP. Different parasites induced IP of different lengths, perhaps reflecting the ability of each parasite to overcome the host defences. Species that induce short IP may either be easily halted in their penetration by the host (indicating a successful defence) or may be especially aggressive, breaking through the end of the IP and entering the host cytoplasm. Long IP, such as those formed in response to Verticillium, probably indicate a h e balance between penetration and wall deposition. The success of the parasite in entering the spore cytoplasm was not assessed in comparison to IP length in this study. FRC were formed both in dead and live spores. When FRC developed in living spores, it appeared that the parasite elicited no visible response by the host. In contrast, the hyphae of species that induced IP immediately stimulated IP formation as the hypha penetrated the inner spore wall. FRC previously have been demonstrated to be induced by three
465 species (Rhizidiomycopsis sfomafosa, Spizellomyces acuminafus, and S. punctatum (Ross & Ruttencutter, 1977; Sparrow, 1977; Schenck & Nicolson, 1977; Tzean, Chu & Su, 1983). although the last species is now thought to be more saprotrophic than parasitic (Paulitz & Menge, 1984). In our study, pathogenic species were more frequently isolated from healthy or mottled spores. Species isolated only from brown or dead spores typically were not pathogenic. These species (e.g. Fusarium sp. Gliomastix sp., Morfierella ramanniana, and Trichoderrna sp.) appear to be saprobes invading already parasitised spores. An exception was Humicola fuscoafra, a parasitic species isolated only from dead spores of G. gigantea. Parasitism of spores of Glornw deserficola (as G. fasciculafum (see Trappe, Bloss & Menge (1984)) by H. fuscoafra was reported by Daniels & Menge (1980). The parasite entered the spores of this fungus through the hyphal attachment and did not form FRC or IP in the spore wall. In our study, hyphae of H. fuscoatra formed abundant FRC in the spores of G . giganfea. No entry through the hyphal attachment was observed. These results suggest that parasitism by fungi and actinomycetes may be an important component of the decline in spore abundance of G. giganfea in sand dunes during spring and summer. This work lends further evidence to support the view that parasitic micro-organisms can significantly reduce the inoculum potential of A M fungi in field soils (Ross & Ruttencutter, 1977; Bagyaraj & Menge, 1978; Daniels, 1981; Paulitz & Menge, 1984, 1986; Hetrick, et al., 1986, 1988; McGonigle & Fitter, 1988; Lee & Koske, 1994). We thank Dr Jane Gemma for many helpful suggestions and advice in the lab work and Dr Roger Goos for taxonomic assistance and encouragement.
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