J. Phytopathology 145, 335-338 (1997)© 1997 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-1785

Ptcmi Pathology Departmenl, University of Florida, Gainesville, FL, USA

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhorniae

Y. M, SHABANA' and R. CHARUDATTAN-'

Authors' address: 'Plant Pathology Department, Faculty of Agriculture, Mansoura University, El-Mansoura, Egypt; ^Plant

Pathology Department, University of Florida, Gainesville, FL, USA

With 4 figures

Received December 30, 1996; accepted March 1, 1997

AbstractPreparation and regeneration of mycelial protoplastsfrom Aliernaria eichhorniae were examined. A com-mercially available muralytic enzyme, Novozym 234, wasused for isolation of protoplasts. The mycelial age andthe pH of the stabilized buffer affected the formationof protoplasts. The maximum production of protoplasts(3,9 X 10*/gfresh weight mycelia) was obtained from 24-h-old mycelia digested with Novozym 234 (20 mg/m!) ina stabilized buffer of pH 6.4 and incubated in the dark at30"C on a rotary shaker (90 r.p.m.) for 6h. Mor-phological characteristics of the protoplasts varied anddepended on the age of the mycelia used in protoplastproduction. Moreover, mycelial age had a highly sig-nificant influence (P = 0.0001) on the frequency of pro-toplast regeneration.

ZusammenfassuDgHersteOung und Regeneration von Alternaria eicMtorniae-Mycel-protoplastenUntersucht wurde die Regeneration von Protoplasten,die aus Alternaria eiehhorniae-Myzel hergestellt wordenwaren. Um die Protoplasten aus dem Myzel herstellen zukonnen, wurde das kaufliche Enzympraparat Novozym234 angewandt. Das Alter des Myzels sowie der pH-Wertdes stabilisierenden Puifers beeinflufite die Pro-toplastenbildung. Die maximale Protoplastenproduktion(3,9 X 107g Myzeifrischgewicht) wurde aus 24 h altemMyzel gewonnen, welches mit Novozym 234 (20 mg/ml)in einem stabilisierenden Puffer bei pH 6 verdaut und fur6h bei 30°C ohne Licht auf einem Kreisschuttler (90Upm) inkubiert wurde. Die morphologischen Merkmaleder Protoplasten waren unterschiedlich und wurdendurch das Alter des verwandten Myzels bestimmt. Daru-berhinaus hatte das Myzelalter einen sehr signifikantenEinfluB (P = 0,0001) auf die Haufigkeit der Pro-toplastenregeneration.

IntroductionFungal protoplasts are useful tools for studying the gen-etics of fungi involving protoplast fusion and trans-formation (Peberdy and Ferenczy, 1985; Peberdy, 1989;Wagner and Wilkinson, 1993). Protoplast fusion has beenused for a variety of purposes in fungal biotechnologyincluding: (1) improved production of antibiotics(MinuthandEsser, 1983), enzymes (Toyamaetal., 1984),and organic acids (Kirimtjra et al., 1986); (2) productionof fruiting bodies such as perithecia (Leslie, 1983) andmushroom sporocarps (Toyomasu and Mori, 1987); (3)production of heterokaryotic mycelium (Leslie, 1983);and (4) intergeneric crosses (Minuth and Esser, 1983;Pina et al., 1986). Protoplast techniques have also pro-vided an understanding of the genetics and biochemistryof plant pathogens, i.e. Pyricularia oryzae (Tanaka et al.,1981), Rhizocionia solani (Hashiba and Yamada, 1984),and Selerotium rolfsii (Deshpande et al., 1987). Inter-specific protoplast fusion between related species hasbeen used to improve several industrially important traitsin Aspergillus (Kirimura et al., 1986), PenieilUum (Anneand Eyssen, 1978), Trichoderma (Toyama et al., 1984),and yeasts (Pina et al., 1986).

This study is part of ongoing efforts to develop a highlyvirulent pathogen to control waterhyacinth [Eiehhorniacrassipes (Mart.) Solms]. A key to this might be the arti-ficial increase in virulence via introducing gene(s) enco-ding for phytotoxin production (Charudattan et al.,1996). The specific objective of this study was thereforeto develop an effective method for protoplast formationand regeneration from the mycelium of Alternaria eich-horniae Nag Raj & Ponnappa, a host specific pathogen ofwaterhyacinth, an aquatic weed of worldwide importance(Shabana et al., 1995a, b).

Materials and MethodsFungus and culture conditionsA. eiehhorniae (isolate no. 5, Ae5) was used. Mycelialsuspension of Ae5 in sterile water was prepared from

US,Copyt,Eh.Cfcanmce Center Code Slatcment: 0931-1785/97/4509-0335 S 14.00/0

336 SHABANA and CHARUDAITAN

hyphal tips of 1-week-old culture grown on potato dex-trose agar (PDA; Difco, Detroit, Ml, USA) medium andused for inoculation of 250-ml Erlenmeyer flasks con-taining 50 ml of potato dextrose broth (PDB; Difco).Inoculated flasks were placed on a rotary shaker (120r.p.m.) and incubated at 30 C in the dark for 24 and 48 h.

Protoplast fonnationNovozym 234 (Novo Industri, Bagsvared, Denmark)solutions were prepared in two stabilized buffers of pHlevels of 8.6 and 6.4 at 20mg/ml. The stabilized bufferswere prepared by adding 0.1 MNa2HPO4 (pH = 8.92)gradually to 0.9MKC1 (pH = 5.85) while checking thepH to obtain the specified values (8.6 and 6.4). Theenzyme solutions were sterilized by filtration through0.2/jm Acrodisc nonpyrogenic filters (Gelman SciencesInc., Ann Arbor, MI, USA).

Twenty four-h-old and 48-h-oid mycelia were har-vested under aseptic conditions with a Buchner funnellined with Whatman no. 1 filter paper using light suction(7-10 psi) and washed twice with stabilized buffer.Washed mycelia, 250 mg wet weight, were suspended in3 mi of Novozym solutions, prepared as mentionedabove, in 25-m! Erlenmeyer fiasks. Flasks were placed inwire baskets on a rotary shaker (90 r.p.m.) for 6 h at 30 Cin the dark. The incubated mixtures were filtered through30-;im-mesh nylon to remove mycelial debris.

Purification of protoplastsTo purify the crude protoplasts, 8 ml of the filtrated lysatewere gently layered over 1 ml of 1.4 M sorbitol solutionand centrifuged at 772 x g for 18 min at 5'C. Protoplastswere recovered in the pellet, and the supernatants(enzyme + sorbitol solutions) were checked micro-scopically to ensure that they were protoplast-free. Theprotoplasts were washed by centrifugation with stabihzedbuffer (pH 6.4). Purified protoplasts were suspended in5 ml of the stabilized buffer and the intact and distortedprotoplasts were counted with a haemacytometer undera microscope. Calculations were made to present theprotoplast counts as per gram of fresh weight mycelia.

Regeneration of protoplastsRegeneration of protoplasts was performed on full- andhalf-strength fresh potato dextrose containing 2% agar(FPDA) [full-strength; potato extract (prepared by boil-ing 200 g of fresh, cut potato in 800 ml of water, andfiltering), 20 g D-glucose, and water to make 1 1] in 9-cm-diameter Petri dishes. The purified protoplast suspensionwas diluted adequately and 0.025 ml of the protoplastsuspension was added per plate. A layer of 0.5% wateragar was also added for a better distribution of the pro-toplasts. Inoculated plates were incubated at 25 C in thedark for 48 h. Seven plates, as replicates, were used foreach medium. The regeneration frequency was shown asthe percentage of the number of colonies formed.

Other cultural conditions for protoplast reversion weretested, namely, using water agar (WA, 2% agar) as arecovery medium for regeneration of protoplasts, wrap-ping plates with one or two layers of Parafilm (American

National Can, Greenwich, CT, USA) vs. unwrapping,and not adding the 0.5% WA layer on the protoplastsuspension.

Statistical analysesThe experiments were repeated at least twice. The dataof protoplast reversion were analysed with the StatisticalAnalysis System (SAS Institute, 1988). All multiple com-parisons were first subjected to analysis of variance(ANOVA) and significant differences among means weredetermined with the Least Significant Means test.

ResultsRelease of protoplasts from the myceliumThe age of the mycelium markedly affected the isolationof protoplasts. Much higher yields of protoplasts wereobtained from the 24-h-old mycelia than from the 48-h-old myceha (3.85 x 10' vs. 3.38 x 10*, respectively)(Table 1). Moreover, the deformation of the protoplastsproduced from 24-h-old mycelia was less than that from48-h-old mycelia (1.3% vs. 3.7%. respectively). The pHof the stabilized buffer used for enzymatic solution wasanother critical factor for effective production of pro-toplasts; the maximum protoplast yield was obtainedwhen a stabilized buffer of 6,4 pH was employed.

Release of spherical cells from the 24- and 48-h-oldmycelium of Ae5 was shown as in Figs 1, 2, 3, and 4. Themorphological characteristics of the resultant protoplastsvaried as the mycelium used in the enzymatic digestionaged. Protoplasts released from 24-h-mycelia were appar-ently smaller in size and non-vacuolus (Fig. 2, 3), whilethose produced from 48-h mycelia were large and vacuo-lated (Fig. 1). The latter frequently contained one or twolarge vacuoles displacing the cytoplasm to one side(Fig. 1).

The use of a distilled-water rinse during the proceduresof protoplast production led to the production of dis-torted and non-spherical protoplasts; thus, stabilizedbuffer had to be used for this purpose.

Regeneration of protoplastsAn ANOVA showed that the age of mycelium used forprotoplast isolation had a high significant effect(P = 0.000!) on the percentage of regeneration of theprotoplasts (Table 2). Thus, protoplasts formed from24-h-old mycelia significantly produced higher reversionfrequencies than those isolated from the 48-h-old mycelia(P < 0.004) (Table 2). The medium and the mediumX mycelial age interaction did not influence the regen-erative capacity of the protoplasts (Table 2; ANOVA). Full-strength and half-strength FPDA were significantly equi-valent as for protoplast reversion.

Very weak reversion of protoplasts occurred on WA;sparse colonial growth with very low regeneration fre-quency were obtained. Dense mycehal growth and highregeneration frequencies were obtained on both full- andhalf-strength FPDA with being full-strength FPDA bestin terms of linear growth and growth density. Regen-erated cells showed colonial growth which were identicalto those of the parent isolate.

Preparation of Aiternaria eichharniac 337

Table IInfiuence ofculture age and hydrogen ion concentralion of the stabilized buffer on the proloplast formation of Aliermiria cichhormae 5 (Ae5)"

Mycelia! age (h)

24244848

pH of stabilized bufler

8.66.48.66.4

Total protoplasts gfresh wt mycelia

4.«xlO-'3.85 X \if'2.4 X 10*3.38 X lO"

Intacl protoplasts gfresh wt myceiiii

4.8 X 10'3.8 X 10'2.4 X iO'2.3 X It)'

Deformedprotoplasts.g fresh wt

mycelia

4.8 X 10"NA

8.4 X 10''

Sphcroplast diameter(fim)''

NM''4.4--13.2 (7.S)

NM4.4-24.2(13.3)

^Conditions for Ihe enzymatic reaction: 250 mg wei myceiium;3 ml enzyme solution (20 mg.m) Novozym in a stabilized buOerK 30'C. 90 rpmrotary shakiiig, 6h: H'alues presented as; range foHowcd b>" means im brackets): 'not applicable because of the low coiinl of protoplasts: ' notmeasured because of the insitinificance of this treatment.

.A....£.Figs 1--4 I, Pro loplas l s frotn the 48-h-old mycelia of Aliernarm eidihormae. 1 \ ;.WU.i. „. |-;ii>-iesee;:- . •.i.-ei. U-S:.!:!!L-I; ^i,-•.•.:,-.•, , .• '.•,•.(•••.,•,:;:•protoplasts, produced from 24-h-old mycelia. under UV lights. A Zeiss fluorescent microscope with an exciter filter BG12 and barrier filter 47(giving transmissions of 330-500 nm and >460nm, respectively) were used, (X 250); 3, Direct production of germ tubes and development ofhyphae from spherical protoplasls isolated from 24-h-old mycelia of Alumaria eichhoriiiac- (X 1000); 4. Production of multiple germ ttibes anddevelopment of hyphae from spherical protoplast regenerated on a half-strength potato de.xtrose medium containing 2° i agar. 24 h after placingthe plate iti a refrigerator at 9 X . (X 1000)

338 SHABANA and CHARUDATTAN

Table 2Influence of two media and culture age on frequency of reversion ofAlternaria eichhorniae 5 (Ae5) protoplasts to prototrophy and lineargrowth of the recovered colonies^

Medium

FPDAFPDAiFPDA'FPDA

Source

ModelMedium (M)

% Colony diameterMycelia! age (h)' Regeneration'^

24482448

df

1Mvcelial age (MA) 1MxMAError

I24

30.86 + 3.13a25.00 +2.78b32.29+1.98a26.29±2.81b

ANOVA

MS F

86.32 n.7912.89 1.76

246.04 33.600.04 0.007.32

(mm)

4-85-93-74-8

P

0.00010.200.00010.94

"Regeneration of protoplasts was performed on full- and half-strengthfresh potato dextrose containing 2% agar (FPDA) and 0.025 ml of anadequately-diluted protoplast suspension was added per plate. A layerof 0.5% water agar was added for a better distribution of the proto-plasts. Plates were incubated at 25'C in the dark for 48 h; ""culture ageused for protoplast formation with ertzyme digestion; 'calculated aspercentage of the number of colonies recovered, total number of pro-toplast plated. Values reported are means + standard de\iation. Valuesfollowed by the same letter are not significantly different according toLS means test (P ^ 0.004).

There was no notable effect of adding a layer of 0.5%WA to the protoplast suspension on reversion frequency.However, this addition resulted in better distribution ofthe regenerated colonies. Also, wrapping plates with oneor two layers of Parafilm had no effect on the frequencyof protoplast regeneration.

DiscussionAlthough mechanical and other non-enzymatic methodsfor protoplast isolation have been reported, most workershave favoured the use of lytic enzymes for protoplastformation, these generally being the most rapid (Peberdy,1979). The components of the lytic digestion are theenzyme, the osmotic stabilizer, and the organism.

The existence of various protoplast sizes resulting fromthe same treatment might be, as described by Gibson andPeberdy (1972), due to the isolation of protoplasts fromdifferent parts of a hypha (tip or older regions). Thisfinding can be further explained by the observation of themorphological variation obtained from different mycehalages suggesting that larger and vacuolus protoplastsrelease from older-age mycelium.

The decrease in the regenerative capacity of the 48-h-old mycelia could be ascribed to the increased amount ofthe deformed protoplasts produced from those myceha.A characteristic pattern observed for the protoplastreversion was that one or more germ tubes were directlyproduced from spherical protoplasts (Fig. 4).

Despite the low frequencies of protoplast regenerationwe obtained, they are better than those reported byothers, namely, Kirimura and Usami (1986) whoachieved 7,2% regeneration frequency from Aspergillusniger with a combined use of several etizymes for pro-

toplast formation and using Czapek's agar medium con-taining 0.7 M KCl for regeneration; and Andrade et al.(1992) who obtained 8% regeneration frequency fromCandida fenniea. However, further studies including basalconditions, combinations and concentrations of lyticenzymes, different osmotic stabihzers, incubation time,etc., should be explored for improving the protoplastreversion.

Acknowledgements

The author thanks the International Foundation for Science, Stock-holm. Sweden, for providing financial support for this research.

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