Cellulolytic and pectinolytic activities ofCapnodium isolates (sooty mould) from Zimbabwe

E. Mwenje and N. Mguni

Abstract: The cellulolytic and pectinolytic activities of fiveCapnodium(sooty mould) isolates previously associatedwith preharvest spoilage of avocado (Persea americanaMill.) fruits in Zimbabwe were assessed in liquid culture andin artificially infected avocado fruits. Polygalacturonase, endo-1,4-β-glucanase, and exo-1,4-β-glucanase activities weredetermined by measuring the increase in reducing groups using the dinitrosalicylic acid method, while for pectin lyaseactivity the thiobarbituric acid method was used. The five isolates showed the ability to produce polygalacturonase,pectin lyase, and endo-1,4-β-glucanase enzymes. Exo-1,4-β-glucanase activity was only detected in infected avocado tis-sue. The greyish blackCapnodiumisolates (Av7, Av8, and Av12) belonging to group I showed higherpolygalacturonase and pectin lyase activities in both liquid culture and infected fruits than isolates Av3 and Av10 fromgroup II. The same isolates showed higher exo-1,4-β-glucanase activity in infected avocado fruits. Results indicate thatCapnodium, which normally is nonpathogenic, secretes cell wall degrading enzymes, especially pectic and cellulase en-zymes. The detection of these enzymes in inoculated avocado fruits suggests a possible role in the preharvest soft rotof avocado fruits caused byCapnodiumin Zimbabwe.

Key words: Capnodium, sooty mould, pectic enzymes, avocados, cellulases.

Résumé: En milieu de culture liquide et sur des fruits d’avocat (Persea americanaMill.) artificiellement infectés, lesauteurs ont évalué les activités cellulolytiques et pectinolytiques de 5 isolats deCapnodium(fumagine) préalablementassocié à une dégradation pré-récolte des fruits d’avocat, au Zimbabwe. Ils ont déterminé les activités de la polygalac-turonase, de l’endo-1,4-$-glucanase et de l’exo-1,4-$-glucanase, en mesurant l’augmentation des groupes réducteurs,par la méthode à l’acide dinitrosalicylique, alors que pour l’activité de la pectine lyase ils ont utilisé la méthode àl’acide thiobarbiturique. Les 5 isolats manifestent la capacité de produire les enzymes polygalacturonase, pectine lyaseet endo-1,4-$-glucanase. On ne détecte l’activité de l’exo-1,4-$-glucanase que dans les tissus d’avocat infectés. Lesisolats gris-noir duCapnodium(Av7, Av8 et Av12) appartenant au groupe I, montre des activités plus élevées de la poly-galacturonase et de la pectine lyase en milieu liquide aussi bien que dans les fruits infectés, que les isolats Av3 et Av 10du groupe II. Ces mêmes isolats montrent une plus forte activité de l’exo-1,4-$-glucanase dans les fruits d’avocat infectés.Les résultats indiquent que leCapnodium, qui normalement n’est pas pathogène, secrète des enzymes dégradant les paroiscellulaires, surtout des pectinases et des cellulases. La détection de ces enzymes dans des fruits d’avocat inoculés suggèreun rôle possible dans la pourriture molle pré-récolte des fruits d’avocats causée par leCapnodium, au Zimbabwe.

Mots clés: Capnodium, fumagine, enzymes pectiques, avocat, cellulases.

[Traduit par la Rédaction] Notes 1495

Introduction

Capnodiumis a sooty mould (dark fungus) that normallyis nonpathogenic and is found on many plant leaf surfaces,as well as on some man-made structures. Sooty moulds arenormally associated with honeydew excreted by sucking in-sects and with plant exudates. Apart fromCapnodium, othersooty moulds include Cladosporium, Aureobasidium,Antemariella, Limacinula, and Scorias (Kessler 1992;Reynolds 1998). In Zimbabwe,Capnodium has been re-corded on avocado (Persea americanaMill.) fruits (Masukaet al. 1998) and has been listed as causing disease on avoca-

dos. However, no description of the disease has been pub-lished. In 1998,Capnodiumwas implicated in the preharvestspoilage of avocado fruits in Zimbabwe (Churu 1999). Twogroups (I, II) of Capnodiumspp. were described based ontheir morphology, medium colouration when grown on po-tato dextrose agar (PDA), and lesion diameter on inoculatedavocado fruits (Churu 1999). Avocado fruits showed soft rotsymptoms, which suggested the involvement of cell wall de-grading enzymes, especially pectic enzymes (Collmer andKeen 1986). The pathogenicity of most fungi causing softrot diseases has implicated their ability to produce cell walldegrading enzymes. The possession of these enzymes doesnot, however, guarantee that an organism is pathogenic orthat the enzymes are involved in disease development. Forexample, Aspergillus nidulans,a saprophytic ascomycete,produced levels of pectic enzymes that were equal to thoseproduced by soft-rotting Erwinia species (Dean andTimberlake 1989). Strong correlative evidence, however,supports the involvement of endopolygalacturonase in dis-eases characterized by soft rotting or tissue maceration

Can. J. Bot.79: 1492–1495 (2001) © 2001 NRC Canada

1492

DOI: 10.1139/cjb-79-12-1492

Received July 10, 2001. Published on the NRC ResearchPress Web site at http://canjbot.nrc.ca on December 18, 2001.

E. Mwenje1 and N. Mguni. Department of Applied Biology& Biochemistry, National University of Science andTechnology, P.O. Box AC 939, Ascot, Bulawayo, Zimbabwe.

1Corresponding author (e-mail: emwenje@nust.ac.zw).

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(Walton and Cervone 1990). Studies by Mwenje and Ride(1999) suggest a role of pectic enzymes, especiallypolygalacturonase (PG), in the root and butt rot diseasescaused byArmillaria. An isolated polygalacturonase (PG1)enzyme fromArmillaria showed the ability to cause cellmaceration and death of cucumber tissue when healthy tis-sue was incubated with the enzyme (Mwenje and Ride1999). The same enzyme was recovered from infected cas-sava tuber tissue that showed signs of soft rot and tissuemaceration (Mwenje et al. 1998).

The cell wall degrading enzymes ofCapnodiumhave notbeen described. The objective of this study was, therefore, todetermine the activities of pectic enzymes and cellulasesboth in liquid culture and in artificially infected avocadofruit tissue.

Materials and methods

Origin and maintenance of isolatesTen isolates ofCapnodiumwere obtained from diseased avocado

fruits in Bulawayo. Diseased avocado fruits showing brown lesionswere collected from the tree and surface sterilized by wiping themwith cotton wool soaked in 70% ethanol. Small sections (0.5 cm)were cut from infected fruits and sterilized in 10% sodiumhypochlorite for 5 min. The infected sections were placed on PDAand incubated at 25°C in the dark. Subcultures were carried out onfresh PDA plates. The isolates were maintained on PDA slants untilrequired. Before isolates could be used in enzyme assays, they werefirst purified on antibiotics. In all experiments, 5-day-old cultureswere used to inoculate flasks or avocado fruits. Three isolates (Av7,Av8, Av12) representingCapnodiumgroup I and two isolates (Av3,Av10) representingCapnodiumgroup II were used in the enzymaticassays and inoculation of avocado tissue.

Preparation and inoculation of liquid mediumCrude cell walls were prepared from Msasa (Brachystegia

spiciformis) segments based on the method described by Mwenjeand Ride (1996). Fresh segments were ground into fine powder.The powder was further treated by soaking in 95% ethanol for 1 hfollowed by filtration and rinsing twice in 95% ethanol for 10 min.The preparation was extracted twice in acetone (10 min each) andthe crude extract was air dried and stored at room temperature. Forpreparation of the liquid medium, 1 g of thecrude cell walls wasadded to 250-mL conical flasks containing 50 mL Vogel’s saltsmedium (Vogel 1956). Four pieces of colonized agar (measuring0.5 × 0.5 cm), taken from a 5-day-old culture of each of the fiveCapnodiumisolates, were used to inoculate the flasks. Three flaskswere used per isolate; these flasks were kept stationary and incu-bated at 25°C for 10 days in the dark.

Inoculation of avocado fruitsAvocado fruits for inoculation were obtained from the market.

Before being wounded, avocados were surface sterilized by wipingthem with 70% ethanol. Six wounds were then made on each avo-cado using a 4-mm cork borer. The wounds were inoculated witheach of the five isolates growing on PDA and then covered withparafilm to reduce mould contamination or drying of inoculum.The inoculated avocados were incubated at room temperature (23–24°C) for 7–10 days. Controls consisted of a wound inoculatedwith uncolonized agar disks.

Enzyme productionLiquid culture filtrate was obtained by filtration using Whatman

No. 1 paper. The culture filtrate was used immediately for enzymeassays or stored in small aliquots below –20°C. For enzyme extrac-

tion from infected tissue, necrotic tissue showing infection was re-moved from avocados with a sterilized scalpel. Infected tissue wasweighed from each point of infection and immediately extracted byhomogenization in 0.01 M sodium chloride. This was followed bycentrifugation at 1800 ×g for 15 min. Filtrate was treated and storedas described for liquid culture filtrate. The filtrates were used forcolometric assays without concentration or dialysis.

Enzyme activity assaysPG activity was assayed by estimating increase in reducing

groups as described by Miller (1959). The enzyme reaction mix-ture contained 100µL (1g/mL of infected avocado tissue) of fil-trate from infected avocado or 200µL liquid culture filtrate, and500µL of 0.5% (w/v) polygalacturonic acid in 0.2 M sodium ace-tate buffer at pH 5.5. Preliminary experiments using healthy avo-cado fruit tissue extracted in the same way as the infected tissueshowed no PG activity in uninfected avocado fruit tissues. Themixture was incubated at 30°C for 1 h, then 800 or 900µL of wa-ter wasadded to the tube to give a volume of 1.5 mL. An equal vol-ume (1.5 mL) of dinitrosalicylic acid was added to all tubes, thenboiled for 15 min in a boiling water bath. Absorbance readingswere taken at 575 nm after the tubes had cooled to room tempera-ture. Controls contained culture filtrates boiled for 30 min. Enzymeactivity was expressed as micrograms galacturonic acid equivalentsproduced per millilitre of culture filtrate per hour.

Pectin lyase (PL) activity was assayed by the thiobarbituric acidmethod as described by Ayers et al. (1966) using 1% pectin as asubstrate. The reaction mixture contained 600µL of culture filtrateand 1% pectin dissolved in Tris buffer. After 1 h incubation at30°C, 1.08 mL of 0.04 M thiobarbituric acid and 0.45 mL of 1 MHCl were added to stop the reactions. Absorbance readings weremeasured at 550 nm and these were taken to indicate PL activity.Boiled culture filtrate was used as a control.

Endo-1,4-β-glucanase (endoglucanase, carboxymethyl cellulase)activity was assayed as described above for PG except that the re-action mixture contained carboxymethylcellulose (0.5% w/v) as asubstrate. A glucose standard was used and enzyme activity ex-pressed as micrograms glucose per millilitre enzyme per hour.Exo-1,4-β-glucanase (exoglucanase) activity was determined by es-timating increase in reducing groups. The reaction mixture con-tained 1 mL of filtrate, and 2 mL of substrate (20 mg crystallinecellulose) in sodium acetate buffer (pH 5.5). For culture filtratefrom infected avocados, 200µL of enzyme was added to 1.5 mL ofthe substrate solution. The reaction tubes were incubated at 30°Cfor 72 h. After incubation, tube contents were filtered to removeundissolved or unhydrolysed substrate. This was followed by mix-ing 1.5 mL of the reaction mixture with 1.5 mL dinitrosalicylicacid and boiling as described before. Absorbance readings weretaken at 575 nm and enzyme activity was expressed as microgramsglucose per millilitre filtrate per hour. The controls contained cul-ture filtrate boiled for 30 min.

The protein concentration of the culture filtrate was determinedusing the Bio-rad reagent as described by Bradford (1976).Absorbance readings were taken at 595 nm within 5 min. Proteinconcentrations were expressed as micrograms protein per millilitreusing bovine serum albumin as a standard (20µg/mL).

Results

PG activityDuring growth in a medium containing 2% cell walls

(w/v) in Vogel’s medium, all theCapnodium isolates se-creted high levels of PG (Fig. 1) as revealed by activity as-says using the dinitrosalicylic acid test for reducing sugars.The PG activities of isolates Av7, Av8, and Av12 belongingto Capnodiumgroup I were higher than those of group II

© 2001 NRC Canada

Notes 1493

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isolates Av3 and Av10 (Fig. 1). The PG activities of the fiveisolates when inoculated onto avocado fruit tissues (Table 1)showed a similar pattern, but higher activity than that ob-

served in liquid culture. The five isolates produced lesionson avocado tissue. There were no lesions produced by thecontrols and no enzyme activity was detected in the controls.

PL activityThe results showed that the ZimbabweanCapnodiumiso-

lates were capable of producing PL in cell wall medium(Fig. 2) and in infected avocado fruits as determined by ac-tivity assays using the thiobarbituric acid method. Therewere differences in PL production among isolates. Isolatesof group I (Av7, Av8, and Av12) exhibited the highest PLactivities, nearly four times higher than those of group IIisolates (Av3 and Av10) (Fig. 2). The pattern of PL produc-tion was the same in liquid medium on cell walls and in in-fected tissue (Fig. 2, Table 1). PL activity was not detectedin the control.

Endo-1,4-β-glucanase activityThe ZimbabweanCapnodiumisolates showed the ability

to produce endo-1,4-β-glucanase activity when carboxy-methyl cellulose was used as a substrate. Group II isolates(Av3 and Av10) showed higher endo-1,4-β-glucanase activitythan group I isolates (Av7, Av8 and Av12). However, thespecific activities of all isolates were similar (Fig. 3). Endo-1,4-β-glucanase activities in infected tissue were also higherfor Av3 and Av10 (group II) isolates (Table 1). Enzyme ac-tivity was not detected in the control.

© 2001 NRC Canada

1494 Can. J. Bot. Vol. 79, 2001

IsolatePG activity(µg galA·mL enzyme–1·h–1)a

PL activity(absorbance)b

Endo-1,4-β-glucanase activity(µg glucose·mL enzyme–1·h–1)

Exo-1,4-β-glucanase activity(µg glucose·mL filtrate–1·h–1)

Av3 1900 0.287 988 1.649Av10 1925 0.260 971 1.085Av7 3738 0.453 767 3.472Av8 3525 0.446 625 3.125Av12 3750 0.464 558 2.561

aPG, polygalacturonase; galA, galacturonic acid.bPL, pectin lyase. Activity measured as absorbance taken at 550 nm.

Table 1. Pectinolytic and cellulolytic activities of fiveCapnodiumisolates from Zimbabwe when the isolates were inocu-lated onto healthy avocado fruit tissue and left for 7 days.

Isolates

PG

activ

ity(µ

g ga

lA·m

Le

nzym

e-1h·

-1)

×10

0

Spe

cificactivity

( µg galA·µg prote

in -1·h-1)

Av3 Av10 Av7 Av8 Av120.0

2.5

5.0

7.5

10.0

12.5

Total activitySpecific activity

0.0

2.5

5.0

7.5

10.0

12.5

Fig. 1. Polygalacturonase (PG) activity of fiveCapnodiumisolatesfrom Zimbabwe isolated from diseased avocado fruits. Isolateswere grown for 10 days in Vogel’s medium containing cell wallsfrom Brachystegia spiciformis.PG activity was estimated by thedinitrosalicylic acid method using polygalacturonic acid as sub-strate in sodium acetate buffer (pH 5.5). galA, galacturonic acid.

Isolates

PL

activ

ity (

abso

rban

ce)

×10 S

pecific activity (absorbance)

Av3 Av10 Av7 Av8 Av120.00

0.01

0.02

0.03

0.04

Total activitySpecific activity

0.00

0.01

0.02

0.03

0.04

0.05

Fig. 2. Pectin lyase (PL) activity of five ZimbabweanCapnodiumwhen isolates were grown for 10 days in Vogel’smedium containingBrachystegiacell walls. PL activity was esti-mated by the thiobarbituric acid method using 1% pectin as asubstrate. Absorbance values were taken to represent PL activity.Absorbance readings were taken at 550 nm.

Isolates

End

o-1,

4--g

luca

nase

act

ivi

tyβ

(g

gluc

ose

mL

enzy

me

h)

µ·

·-1

-1

Specific activity

(g

glucoseg

proteinh

µµ·

·)

-1-1

Av3 Av10 Av7 Av8 Av120

1

2

Total activity

Specific activity

0

1

2

Fig. 3. Endo-1,4-β-glucanase ofCapnodiumisolates when theisolates were grown for 10 days in Vogel’s medium amendedwith Brachystegiacell walls. Activity was estimated by thedinitrosalicylic acid method using carboxymethyl cellulose as asubstrate in sodium acetate buffer (pH 5.5).

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© 2001 NRC Canada

Notes 1495

Exo-1,4-β-glucanase activityExo-1,4-β-glucanase activity using microcrystalline cellu-

lose as a substrate showed that all isolates were capable ofthis activity. Isolates of group I (Av7, Av8, and Av12) hadhigher activities (Table 1). However, exo-1,4-β-glucanase ac-tivity was not detected in liquid culture filtrate.

Discussion

The ability ofCapnodiumisolated from Zimbabwe to pro-duce cell wall degrading enzymes both in liquid culture andin inoculated avocado fruits was demonstrated. The presentobservations suggest that this normally nonpathogenic sootymould could cause diseases in situations where the plant’sresistance has been lowered by environmental factors suchas drought. This is due to the ability of the mould to producePG, PL, and a combination of endo-1,4-β-glucanase and therare exo-1,4-β-glucanase, which are believed to enable apathogen to penetrate the host’s tissues (Osagie andObuekwe 1991; Isaac 1992). In pathogenesis, exo-1,4-β-glucanase is believed to act synergistically with endo-1,4-β-glucanase for more efficient and systematic degradation andutilization of cellulose (Umezurike 1970).

Zimbabwe has had recurrent drought periods since 1990.This may have lowered the resistance of some plants to dis-eases, thus giving way to opportunistic infections. TheCapnodiumisolates that caused the greatest necrotic lesions(Churu 1999) also showed the highest pectinolytic activitiesin our study. Many studies show that there is a correlationbetween the production of pectic enzymes and the pathoge-nicity of the microorganisms, especially in soft rot diseases(Isaac 1992). Mwenje and Ride (1996, 1997) showed thatthe Armillaria isolates producing high amounts of pectic en-zymes were the most pathogenic. Johansson (1988) alsoshowed that P strains ofHeterobasidion annosum, whichwere more aggressive than S strains, showed high pectolyticactivities both in vivo and in vitro.

In the present study, enzyme activities were higher in vivothan in vitro and exo-1,4-β-glucanase was only produced invivo (infected avocado fruits). This could be due to stimula-tion of enzyme production by the host (avocado) cell wallsas compared with theBrachystegia spiciformis(non-host)cell walls that were used in liquid culture. Although the abil-ity to secrete cell wall degrading enzymes may not guaranteetheir involvement in pathogenicity, the correlation betweensoft rots and enzyme activity suggests the possible role ofthese enzymes in soft rot diseases of avocado fruits causedby Capnodiumspp. The results of this study indicate thatCapnodium, now found to be a pathogen of avocado fruits inZimbabwe, has high pectinolytic and cellulolytic activities.The ability of this mould to secrete these enzymes may ren-der it a potentially serious pathogen to the avocado industryin Zimbabwe.

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