Mycol. Res. 96 (12): 1071-1076 (1992) Prinled in Greal Brilain 1071

Quantification of the nuclear DNA content of two arbuscularmycorrhizal fungi

V. BIANCIOTTO AND P. BONFANTE

Dipartimento di Biologia Vegetale del/'Universita and Centro di Studio suI/a Micalogia del Terreno del CNR, Viale Mattioli 25 1-10125 Torino,Italy

The nuclei of two arbuscular mycorrhizal fungi, Gigaspora margarita and Glomus versiforme, were examined cytofluometrically to (i)determine their DNA amount and (ii) look for differences in their genome size during some steps of their life cycle.

The fluorescence characteristics of DNA-DAP! complex revealed that DNA amount is significantly different. This valueexpressed as arbitrary units and then quantified by using a biological standard is of 0'25--0'27 pg per nucleus in G. versiforme and0'74--0'77 pg per nucleus in G. margarita. There were no differences between their individual spore and hyphal phases.

Genome size and its modifications are important clues to theevolutionary relationships between living organisms, as wellas their cell cycle (Alberts et ai., 1989). Fungi are usuallyconsidered to possess a low amount of DNA in their nucleirelative to higher eukaryotes (Cavalier-Smith, 1985). Theinformation available mostly deals with saprotrophic species,though numerous recent papers present DNA values forfungal plant pathogens (Francis et al., 1990; Whittaker et a!.1991 and references). Specific steps of the fungal life cycle areincluded such as the appressorium formation of Uromycesappendiculatus (Kwon & Hoch, 1991), or the formation ofresistant bodies in anaerobic fungi, such as a Chytridiomycetesisolated from the rumen of a cow (Wubah et al., 1991).

Many of these values have been obtained by cyto­fluorimetry. This technique has not yet been used forarbuscular mycorrhizal (AM) fungi. These are WidespreadZygomycetes that colonize the roots of about 80% of the landplants as mutualistic symbionts (Harley, 1989). They can notyet be studied in pure cultures, however, and little is knownabout their cell biology (Burggraaf & Beringer, 1989) ornuclear status. As expected from their taxonomic position,they are coenocytic organisms with from 9000 nuclei perspore in Glomus caledonium to 20000 in Gigaspora margarita,and 35000 in G. decipiens (Burggraaf & Beringer, 1989; Viera& Glenn, 1990). The hyphae of the intraradical phase aremultinucleate too (Bonfante et al., 1987, Cooke et al., 1987).

However, no comparison has yet been made of nuclear DNAduring the spore phase versus the symbiotic intraradical phase.

The paper therefore reports the DNA content of Gigasporamargarita Becker & Hall and Glomus versiforme Karsten(Berch) and verifies whether differences exist in the genomesize of these two AM fungi during three steps of their lifecycle: spores, extramatrical and intraradical mycelium.

MATERIALS AND METHODS

Fungus and plant material

Spores of Gigaspora margarita (HC/Fungi EI0) were recoveredfrom pot cultures of Trifolium repens L. (clover) by wet sieving(Gerdemann, 1963). Fruitbodies of Glomus versiforme (HC/Fungi EOl) were collected from the surface of 2-month-oldquartz-sand pot cultures of Allium porrum L. (leek) and purifiedby density gradient centrifugation (Furlan et al., 1980). Theisolated spores were surface sterilized with 4 % Chloramine Tand 300 ppm of streptomycin for 20 min, and rinsed withsterile distilled water.

Seeds of Allium porrum L cv. Monstruoso di Carentan andTrifolium repens L. were sown in sterilized quartz sand. Thegerminated seedlings were watered three times a week with alow phosphorus Long Ashton solution (Hewitt, 1966).Mycorrhizal plants were obtained by injecting a sporesuspension obtained from G. versiforme fruitbodies around theleek seeds and single G. margarita spores around the cloverseeds.

Preparation and staining of spore and hyphal nuclei

Fifty spores of G. margarita (350 \lm diam.) and 2000 sporesof G. versiforme (80 \lm diam.) were fixed in 4 % p-form­aldehyde (wIv) in Tris buffer (10 mM Tris (hydroxy­methyl)aminomethane, 10 mM Na-EDTA, 100 mM NaC!,pH 7'4+0'1% Triton X-I00) for 2 h on ice and then washedtwice in the buffer.

The nuclei were isolated by crushing the fixed spores witha glass rod in buffer solution. The cytoplasmic suspension waspassed through two 15 and 5 \lm pore nylon filters: nuclei

The nuclear DNA content of two fungi 1072

Figs 1-3. Nuclear distribution in AM fungi after DAPI staining. Fig. 1. Gigaspora margarita chlamydospore showing a layer of nucleialong the inner spore wall (scale bar = 15 j.lm). Fig. 2. Gigaspora margarita hypha from extraradical mycelium with large nuclei.Mitochondria and/or Bios are stained (arrow) (scale bar = 10 j.lm). Fig, 3. Frozen section of Allium porrum infected by Glomus versiformeshoWing nuclei (arrowhead) in the intercellular hyphae (ih). Arbuscules (A) nuclei are barely recognisable (arrow). Note the large size ofthe host nuclei (hN) for a comparison (scale bar = 20 j.lm).

were recovered and immediately used. All the operations werecarried out on ice.

Nuclear suspension was stained in the dark with 4',6­diamidino-2-phenylindol (DAPI, Sigma) dissolved in Trisbuffer to a final saturating concentration (5'6 j.lM). After 15min of staining, nuclear suspensions were directly preparedon the slides for static cytofluorimetry.

Extramatrical mycelium from roots colonized byG. margarita and G. versiforme was fixed and stained withDAPI as already described.

To measure the intraradical mycelium, root segments ofleek colonized by G. versiforme were fixed in 4 % p­formaldehyde in 50 mM PIPES,S mM MgS04, 5 mM EGTA(MTSB) pH 6'9 for 2 h, washed in MTSB pH 6'9 and leftseveral days in a solution consisting of 0'5 % p-formaldehydeand 1'5 M sucrose in MTSB.

Thick frozen sections (20 j.lm) obtained with a cryo­microtome (Micron-Heidelberg) were rinsed with Tris buffer

pH 7'4 and stained with DAPI as already described. Thediameters of the spore and hyphal nuclei were measured afterDAPI staining under uv light with a Zeiss Universalfluorescence microscope with a 100 x objective.

Static cytofluorimetry

The fluorescence intensity of DAPI stained nuclei wasmeasured with a Leitz Diaplan microscope equipped with aMPV microscope compact photometer using an Epson PX 4

computer and an incident light illuminator Leitz employing a100 W mercury lamp. The excitation filter was a BP 340-380,dichroic mirror RKP 400 and barrier filter LP 430. Thephotomultiplier was operated at a constant voltage (600 V);with a FL 40/0.70 objective; measuring spot was 0'2 mm indiameter, while the field diaphragm was slightly larger. Thebackground fluorescence was directly measured and subtractedfrom the fluorescence of the nuclei by the computer. The

V. Bianciotto and P. Bonfante 1073

following precautions were taken to minimize the quick fadingfluorescence: excitation was reduced to the period duringwhich nuclei were centred and focused; only nuclei exposedfor less 10 s were measured; only one nucleus was measuredin each field of view exposed to uv.

Nuclei of the arbuscular hyphae were not measured, due tothe impossibility of discriminating the single fungus nucleusfrom the fluorescence background of the host nucleus.

Genome size

RESULTS

Nuclear visualization and measurement

Table 1. Size of nuclei (mean diam. (~m) ± S.D.) of spore. extraradical andintercellular mycelium'

, 100 nuclei were measured for each sample.Values of each column and not sharing the same letter are significantly

different according to ANOVA one-way test (P = 0'05).

DISCUSSION

Measurement of DNA-DAPI fluorescence as a tool infungal cell biology

The nuclear diameters and DNA-DAPI fluorescence measure­ments revealed marked differences between the genome sizesof these two AM fungi. The ratio between the fungus andCRBC nucleus fluorescence intensity was significantly differentand 65 % difference in G. margarita and G. versiforme DNAcontent was determined. Duran & Gray (1989) have suggestedthat microfluorimetry could be of assistance in fungustaxonomy following their quantification of DNA in 70 smutfungi and two Neurospora species. Even if generalizationshould be avoided until new data on different species areobtained, our results support this statement. since thedifferences found between the two AM species closely fitMorton & Benny's (1990) Glomales classification. Accordingto these authors, Glomus belongs to the suborder Glomineae,along with almost all AM fungi. By contrast. Gigaspora formsa new suborder, Gigasporineae with two genera, Scutellospora

and Gigaspora.In addition to the characterization of strains within the

same species (Meixner & Bresinsky, 1988; Motta et al., 1986),DNA quantification can also assist in the study of ploidy

These results show that (0 Glomus versiforme and Gigasporamargarita possess nuclei with significantly different diameters;(ii) nuclear size changes depending on the step of the life cycle(saprotrophic phase versus symbiotic phase), decreasing fromthe spore to the extramatrical and intraradical mycelium; and(iii) the difference in DNA quantity is significant between thetwo fungi, but not between the two phases of their individualcell cycle.

Genome size was determined by measuring chicken erythro­cytes as the biological standard at the same time of the fungus(Fig.5).

In G. margarita the ratio between the arbitrary fluorescencevalue and that of the standard was 3'0 in sporal nuclei and 3'1in extramatrical hypha!. Measurements were not sought forintraradical mycelium, due to the substantial autofluorescenceof the host plant under uv illumination. In G. versiforme theratio was 8'8 in spore nuclei, 9'1 in extraradical hyphal nucleiand 8'6 in intercellular hyphal nuclei. Since chicken erythrocytespossess 2'3 pg DNA per nucleus, and the fluorescence of theDNA-DAPI complex is stoichiometrically related to theamount of DNA, the genome size can be quantified as shownin Table 2.

Significant differences were found between the two species(0'25-0'27 pg per nucleus in G. versiforme and 0'74-0'77 pgper nucleus in G. margarita), but not between the phases oftheir life cycle.

4'23±0'41a3'75±0'48b3'51±0'43c

G. "ersifvrme G. margarita

2'36±0'24a2'20±0'33b2'06±0'21c

SporeExtraradical myceliumIntercellular mycelium

Nuclei were easily observable after DAPI staining. They weremostly located near the wall of the spore of G. margarita (Fig.1), whereas they were surrounded by the lipid globules inG. versiforme. Both nuclear populations were round and smallin size, though there were substantial differences in theirnuclear diam.: G. margarita, 4'23 ~m (S.D. = ±0'41);

G. versiforme, 2'36 ~m (S.D. = ± 0'24) (Table 1). In theextramatrical mycelium, nuclei were irregular in distributionand shape. Nuclei were numerous and very close in somehyphae (Fig. 2).

Nuclei also changed their shape and size during theintraradical phase. They were oval or roundish in theintercellular hyphae (Fig. 3), but hardly recognizable in thearbuscular branches, where they were smaller than those ofspores and extramatrical hyphae (Balestrini et aI., 1992).

Table 1 summarizes all the results. It can be seen thatnuclear size decreases from the spores to the extraradicalmycelium in both species.

Measurements of the fluorescent DNA-DAPI complex

No differences were noted between spore and extraradicalnuclei of G. margarita and between spore and hyphal nuclei ofG. versiforme (Table 2). The mean fluorescence values inarbitrary units were 54'1 and 55'0 for spore and hyphal nucleiin G. margarita and 17'6, 17'5 and 19'3 for the three steps inG. versiforme. By contrast, differences exist between the twospecies. Histograms reveal a unimodal distribution pattern inall samples: only a few nuclei show a double fluorescencevalue (Fig. 4).

Biological standard

Chicken red blood cells (CRBC) were included in the samplesas internal biological standard to control instrumental andstaining variability (Sgorbati et al., 1986). They were frozen inliquid nitrogen, stored at - 20°C, and defrosted to bemeasured with the fungus nuclei. We assumed 2'3 pg pernucleus as the amount of DNA per red cell (Galbraith et al.,1983; Sgorbati et al., 1986; De Rocher et al., 1990).

68 MYC 96

The nuclear DNA content of two AM fungi 1074

50Glomus rersiforml!

uclei in the pore

.i =54.1

Gigaspora margaritaNuclei in the spore

20 30 40 50 60 70 80 90 100Fluorescence inten ity (arbitrary units)

50

40

'0U" 30c:.....0

.820E

"Z10

00 10

3530

x=17·6S.D. =± 4·7S.E. =± 0·5

25201510

Glomus "ersiforml!uclei in the extra radical mycelium

Fluorescence inten ity (arbitrary unit)5o

0+----,.l<=<.<1

30

'0 x=17·5u S.D. =±4·5" S.E. =± 0·5t:..... 200

.8E"Z

10

10

40

'0ug 30.....o

.8E 20"Z

o 5 10 15 20 25 30 35

Fluorescence intensity (arbitrary unit)

Glomus l'ersiforml!uclei in the intercellular mycelium

30

.t =19·3.~ S.D. =± 7·8u

S.E. =± 0·9"t: 20.....0

.8E"Z

10

o

50

40

'0U"c: 30.....0...~E 20"Z

10

0

Gigaspora margaritaNuclei in the extraradical mycelium

o 5 10 15 20 25 30 35 0 10 20 30 40 50 60 70 80 90 100

Fluorescence intensity (arbitrary unit) Fluorescence intensity (arbitrary units)

Fig. 4. Representative histograms of distribution of DAPI-DNA in arbitrary units during the steps of life cycle of G. versiforrne andG. margarita.

levels through a cell cycle. Our results show that the two AMfungi possess nuclei with a homogeneous unimodal dis­tribution, pointing to a single ploidy level in both spore andsymbiotic phases. Unlike some pathogenic fungi, where hostcolonization depends on the establishment of a diploid phase(for example, Ustilago maydis, according to Wangemann­Budde & Schauz 1991), intraradical hyphae of G. versiformeretain their single ploidy. However, unimodal distribution

does not mean an absence of DNA duplication during hyphalgrowth. According to Tooley & Therrien (1987), it can berelated to a relatively low number of measured nuclei.

The change in nuclear size versus the homogeneous relativeDNA content during their life cycle suggests that the twoAM fungi do not lose DNA during infection, but onlyundergo a change in the chromatin organization. Ultra­structural observations have shown nuclei with dispersed

V. Bianciotto and P. Bonfante 1075

Fluorescence intensity (arbitrary units)

Fig. 5. Histogram of distribution of DAPI-DNA in arbitrary units

in spore nuclei of G. versiforme and chicken red blood nuclei (CRBC)used as internal standard. The ratio between mean CRBC fluorescence

and mean G. versiforme spore fluorescence was 8'8.

40

30.iiiU:>c...0... 20~E:>Z

10

G. versiformespore nuclei

CRBCstandard

agree with the 0'52 pg per nucleus for Phytophthora infestans(Mexican isolates) by Tooley & Therrien (1987). Therefore,the few fungi examined lie between bacteria and highereukaryotes (Cavalier-Smith, 1985).

The two AM fungi can be compared with some obligatebiotrophs, such as Erysiphe graminis, whose DNA amount pernucleus is 0'63 pg (Cavalier Smith, 1985). Viera & Glenn(1990), on the other hand, have reported 3'4 pg per nucleusfor G. versiforme and 1'7 pg per nucleus for Scutellosporapersica. Their figures, however, express a ratio between thetotal sporal DNA and the hypothetical number of nuclei perspore. The advantage of our in situ technique compared witha total extraction is that it measures nuclear DNA only, andavoids other sources, e.g. mitochondria or BIos (Scannerini &

Bonfante, 1991).

This research was supported by National Research of Italy,Special Project RAISA, Subproject no. 2, paper no. 372.

REFERENCESTable 2. Fluorescence measurements of DNA-DAPI (m = mean inarbitrary units)

Genome sizeNumber (m) fluorescence Mean nuclei (pg per

Fungus of nuclei (X±S.D.) Mean CRBC nucleus)

G. versiformeSpore 82 17'6±4'7 8'8 0'26CRBC 30 154'8± 13'3Extraradical 78 17'5±4'5 9'1 0'25

myceliumCRBC 30 159'7±12'6Intercellular 72 19'3 ± 7'8 8'6 0'27

myceliumCRBC 30 166'1± 14'9

G. margaritaSpore 88 54'1 ± 10'5 3'0 0'77CRBC 30 162'3 ± 13"7Extraradical 92 55'0± 14'1 3'1 0'74

myceliumCRBC 30 170'5±6'7

chromatin, but more often with condensed chromatin patches:this could cause a shrinkage in the nuclear volume (Balestriniet al., 1992).

Genome size: arbitrary units and absolute values

Since fluorochrome DAPI binds selectively to A-T pairs,DNA-DAPI fluorescence is proportional to DNA contentwhen DNA of the same base composition is compared(Coleman et a/., 1981; Whittaker et a/., 1991). The ATcomposition of AM fungi is not known, though that of theZygomycetes ranges from 52 to 62 % (Stork, 1966). CRBCshave an AT amount of about 58% (McCarthy, 1969) and canthus probably be compared with AM fungi. This enablesgenome size to be calculated in absolute values.

The DNA amount per nucleus is 0'74-0'77 pg in G.margarita and 0'25-0'27 pg in G. versiforme. These values aremuch lower than in other eukaryotes (Alberts et al., 1989), but

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(Accepted 20 May 1992)

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