Use of response surface methodology to examine chitinase regulation in the basidiomycete Moniliophthora perniciosa

m y c o l o g i c a l r e s e a r c h 1 1 2 ( 2 0 0 8 ) 3 9 9 – 4 0 6

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Use of response surface methodology to examine chitinaseregulation in the basidiomycete Moniliophthora perniciosa

Maıza Alves LOPESa,b, Dayane Santos GOMESa, Maria Gabriela BELLO KOBLITZb,Carlos Priminho PIROVANIa, Julio CEZAR DE MATTOS CASCARDOa, AristotelesGOES-NETOb, Fabienne MICHELIa,c,*aLaboratorio de Genomica e Expressao Genica, DCB, Universidade Estadual de Santa Cruz, Rodovia Ilheus-Itabuna,

Km 16, 45650-000 Ilheus-BA, BrazilbLaboratorio de Pesquisa em Microbiologia, DCBio, Universidade Estadual de Feira de Santana, Km 3, BR 116 (norte),

44031-460 Feira de Santana-BA, BrazilcUMR DAP, Cirad, Avenue Agropolis TA80/02, 34398 Montpellier cedex 5, France

a r t i c l e i n f o

Article history:

Received 26 January 2007

Received in revised form

11 September 2007

Accepted 31 October 2007

Corresponding Editor:

Gareth W. Griffith

Keywords:

Basidiomycota

Central composite design

Chitinase

Mycelial growth

Secretion

* Corresponding author. Laboratorio de GeItabuna, Km 16, 45650-000 Ilheus-BA, Brasil.

E-mail address: [email protected]/$ – see front matter ª 2007 The Bdoi:10.1016/j.mycres.2007.10.017

a b s t r a c t

We report here the first analysis of chitinase regulation in Moniliophthora perniciosa, the

causal agent of the witches’ broom disease of cacao. A multivariate statistical approach

was employed to evaluate the effect of several variables, including carbon and nitrogen

sources and cultivation time, on M. perniciosa non-secreted (detected in mycelium, i.e. in

symplasm and cell wall) and secreted (detected in the culture medium) chitinase activities.

Non-secreted chitinase activity was enhanced by peptone and chitin and repressed by

glucose. Chitinase secretion was increased by yeast extract alone or in combination with

other nitrogen sources, and by N-acetylglucosamine, and repressed in presence of chitin.

The best cultivation times for non-secreted and secreted chitinase activities were 30 and

20 d, respectively. However, chitinase activity was always higher in the mycelium than

in the culture medium, suggesting a relatively poor chitinase secretion activity. Conversely,

higher mycelial growth was observed when the activity of the non-secreted chitinase was

at its lowest, i.e. when the fungus was grown on glucose and yeast extract as sources of

carbon and nitrogen, respectively. Conversely, the induction of non-secreted chitinase

activity by chitin decreased the mycelium growth. These results suggest that the culture

medium, by the induction or repression of chitinases, affected the hyphal growth. Thus,

as an essential component of M. perniciosa growth, chitinases may be a potential target

for strategies to control disease.

ª 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Chitinases (EC 3.2.1.14), belonging to the family of glycosyl

hydrolases, catalyse the hydrolysis of chitin, a linear homo-

polymer of b-1,4-linked N-acetyl-D-glucosamine (NAG)

nomica e Expressao Gen

rritish Mycological Society

residues. Chitinases play an important physiological and

ecological role in ecosystems as recyclers of chitin by generat-

ing carbon and nitrogen sources (Cohen-Kupiec & Chet 1998).

Chitinase production occurs naturally in a wide variety of

organisms, such as microorganisms containing chitin,

ica, DCB, Universidade Estadual de Santa Cruz, Rodovia Ilheus-

. Published by Elsevier Ltd. All rights reserved.

400 M. A. Lopes et al.

bacteria, arthropods, vertebrates, and plants. The physiologi-

cal function of chitinases depends on their source. In bacteria,

chitinases play a role in nutrition by degrading chitin, which is

used as a source of both carbon and nitrogen by the cell

(Wiwat et al. 2002). In plants, most chitinases are induced by

stress factors, such as infection by pathogens containing

chitin, and are considered pathogenesis-related proteins

(Hamel & Bellemare 1995; Krishnaveni et al. 1999; Regalado

et al. 2000). In organisms containing chitin in their cell wall

or in other defined structures, such as in fungi, chitinases

are known to be involved in spore germination, hyphal growth

and branching, development of mycelium and reproductive

structures, cell separation, autolysis, and parasitism (Chet

et al. 1997; De Marco et al. 2000; Flach et al. 1992; Gooday

1990, 1997; Kuranda & Robbins 1991; Zellinger et al. 1999).

Production of microbial chitinases has received worldwide

attention in both industrial and scientific communities,

mainly because of its wide spectrum of applications. Chitin

has a broad range of applications in biochemical, food, and

various chemical industries: it has antimicrobial, anticholes-

terol, and antitumour activities. It is also used in wastewater

treatment, drug delivery, wound healing, and dietary fibre

(Dahiya et al. 2006 for review). Previous studies have investi-

gated substrate type in order to optimize chitinase production;

these have involved several chitinases-producing microor-

ganisms, such as Verticillium lecanii (Liu et al. 2003), Trichoderma

harzianum (Nampoothiri et al. 2004), Streptomyces sp. (Nawani &

Kapadnis 2005), and Penicillium chrysogenum (Patidar et al.

2005). The concept of response surface methodology (RSM)

has eased development processes and has been of significant

use in industry. Recent studies have indicated the use of RSM

for analysing the effects of different factors on proteolytic ac-

tivity (Gobbetti et al. 1999) and optimizing enzyme production

(Nawani & Kapadnis 2005; Souza et al. 1999). However, the reg-

ulation and capacity for chitinase secretion by basidiomycetes

under different culture conditions is, to our knowledge, still

unknown. For this reason we investigated chitinase regulation

in Moniliophthora perniciosa syn. Crinipellis perniciosa; (Aime &

Phillips-Mora 2005), the causal agent of the witches’ broom

disease, one of the most important diseases of cacao in the

western hemisphere (Purdy & Schmidt 1996; Rocha et al.

1993). The disease shows two distinct stages: a biotrophic

and a necrotrophic/saprotrophic phase. In the biotrophic

phase, the fungus causes hypertrophy and hyperplasia of

the tissues, loss of apical dominance, and proliferation of ax-

illary shoots, which results in the formation of abnormal

Table 1 – Variables for screening the five-level central compos

Variable �1.68 �1

Carbon source Chitin Chitin (0.5 %)/

glucose (0.5 %)

Nitrogen

source

NH4H2PO4 Yeast extract

(0.5 %)/NH4H2PO4

(0.1 %)

Incubation

time (days)

10 14

stems (green broom). In the second stage, the fungus changes

to the saprotrophic phase and causes necrosis and death of

infected tissues distal from the original infection site, produc-

ing a dry broom (Ceita et al. 2007; Garcia et al. 2007). Basidio-

mata production and spore formation occur on infected

necrotic tissue (Silva et al. 2002; Scarpari et al. 2005).

In order to evaluate M. perniciosa as an industrial source of

chitinases, as well as to explore the possibility of targeting chi-

tinases for disease control, the present study evaluated, using

the concept of RSM, the influence of different carbon and

nitrogen sources and the cultivation time on the activity of

non-secreted or secreted chitinases from the fungus.

Materials and methods

Culture conditions

Moniliophthora perniciosa strain ALF553 (CCBM000257, UEFS,

Feira de Santana, Brazil) was grown on agar containing

10 g l�1 glucose, 1 g l�1 NH4H2PO4, 0.2 g l�1 KCl, 0.2 g l�1

MgSO4.7H2O, 5 g l�1 yeast extract, 1 g l�1 CuSO4.5H2O and 1 g l�1

ZnSO4.7 H2O at 25 �C for 15 d prior to its use for inoculum

development for chitinase production. Liquid culture medium

containing 10 g l�1 carbon source, 6 g l�1 nitrogen source, 0.2 g

l�1 KCl, 0.2 g l�1 MgSO4.7H2O, 1 g l�1 CuSO4.5H2O, and 1 g l�1

ZnSO4.7H2O was used for inoculum development and for

chitinase production. A 1 cm diam disc of fungal mycelium

was taken from the solid culture, transferred to liquid

medium, and incubated at 25 �C without agitation, for

10–30 d, as described in the experimental design.

Experimental design

A three-variable, five-level central composite design (CCD)

leading to 17 sets of experiments (assays), performed in dupli-

cate, was used to verify the best culture medium conditions to

induce the production of chitinases, as well as to analyse the

mycelium growth. The independent variables consisted of

different incubation times and sources of carbon and nitrogen

(Table 1). The results (dependent variables) were the mea-

sured chitinase activity in the mycelium (non-secreted pro-

teins, i.e. symplastic and cell wall proteins) and in the

culture medium (secreted proteins) and the measured myce-

lium growth. The liquid culture medium used was the same

as described above. The incubation was carried out at 25 �C

ite design

0 þ1 þ1.68

Glucose N-acetyl

glucosamine (0.5 %)/

glucose (0.5 %)

N-acetyl

glucosamine

Yeast extract Peptone (0.5 %)/

yeast extract

(0.1 %)

Peptone

20 26 30

Chitinase regulation in Moniliophthora perniciosa 401

without agitation. The software STATISTICA 6.0 (StatSoft,

Tulsa, OK) was used to generate the design matrix and to

analyse the results. Table 2 presents the design matrix and re-

sponses (results) obtained for chitinase activity in the myce-

lium and in the culture medium, and for the mycelium

growth.

Protein extraction and quantification

Mycelium grown on liquid medium was collected, washed

three times with 35 ml cold Milli-Q water (Millipore, Billerica,

MA), dried on filter paper, and ground in liquid nitrogen. The

powder was transferred to a 2 ml tube, and to each 100 mg

of powder, 1.25 ml of 10 mM phosphate buffer (pH 6) contain-

ing 1 % ascorbic acid was added. The tubes were vortexed

and centrifuged at 20,000 g for 10 min at 4 �C. The superna-

tant, containing the mycelium proteins, was collected and

stored at 4 �C. The culture medium, on which Moniliophthora

perniciosa was grown, was filtered on Miracloth (Calbiochem,

San Diego, CA). The filtrate, containing the secreted proteins

from M. perniciosa, was collected and stored at 4 �C. The con-

centration of the proteins from both the mycelium extract

and culture medium was estimated by a based Bradford assay

(Biorad, Hercules, CA) using bovine serum albumin (BSA) as

the standard protein.

Determination of the chitinase activity

Colorimetric assays of chitinase activity were performed in

triplicate using the purple dye-labelled biopolymeric sub-

strate, CM-chitin-RBV (Loewe Biochemical, Saulerlach,

Germany). Two hundred microlitres of CM-chitin-RBV

(2 mg ml�1) were mixed with 300 ml mycelium extract or cul-

ture medium and 300 ml of 50 mM acetate buffer (pH 5). The

Table 2 – Experimental design and results of the central comp

Assay Variable

Carbonsource

Nitrogensource

Incubationtime

1 þ1 þ1 þ1

2 þ1 þ1 �1

3 þ1 �1 þ1

4 þ1 �1 �1

5 �1 þ1 þ1

6 �1 þ1 �1

7 �1 �1 þ1

8 �1 �1 �1

9 þ1.68 0 0

10 �1.68 0 0

11 0 þ1.68 0

12 0 �1.68 0

13 0 0 þ1.68

14 0 0 �1.68

15 0 0 0

16 0 0 0

17 0 0 0

Experiments were performed in triplicate.

mixture was incubated at 37 �C for 3 h. The reaction was

stopped by the addition of 200 ml of 2 M HCl. Samples were

cooled on ice for 15 min, then centrifuged at 20,000 g for

10 min to remove the non-degraded substrate. The superna-

tant was collected and the assay was performed spectrophoto-

metrically at 550 nm (Cary� 100 UV–visible spectrophotometer;

Varian, Palo Alto, CA). Chitinase activity was measured in

units per nanogram of protein per hour. One unit of chitinase

activity corresponded to an increase in absorbance of 0.1

(Ramırez et al. 2004).

Determination of the mycelium growth

The mycelium growth was obtained by quantifying the myce-

lium mass. Three replicates of the Moniliophthora perniciosa my-

celium disc for each assay were collected, quickly dried on

filter paper, and separated from the medium layer using a razor

blade. The mycelium was weighed after 4 h drying at 60 �C.

Results

Central composite design

Based on the identification of variables by the three-level frac-

tional factorial, a CCD was developed for variables that signif-

icantly affected chitinase activity in the mycelium and in the

culture medium. Table 2 lists the coded values of the levels

of the variables selected in the CCD, as well as the responses

generated by the CCD, such as chitinase activity in the myce-

lium and in the culture medium, and mycelium growth.

A quadratic type model was generated, and all the effects

were considered significant (P< 0.05). The model has a relatively

osite design

Chitinase activity(UA ng�1 protein h�2)

Mycelium dryweight (mg)

Mycelium Culture medium

100.22 11.09 99

93.99 26.22 109

74.13 11.61 116

78.1 16.04 60

161.21 3.03 90

76.77 4.3 115

84.48 12.84 92

51.86 0.451 116

52.53 52.14 29

189.61 4.06 29

47.5 8.37 116

0 0 0

60.6 24.29 159

39.29 2.85 57

61.81 34.28 208

59.87 13.15 206

63.5 32.5 201

402 M. A. Lopes et al.

high R2 value (0.771 for the mycelium, 0.766 for the culture me-

dium and 0.789 for the mycelium growth), indicating that 77,

76, and 79 %, respectively, of the variations between the

assays were due to the differences between the tested culture

conditions. Although the F-value was not predictive, it could

be considered significant at a confidence level of 80 %, mean-

ing that the model is descriptive of the data obtained. The pure

error and the lack-of-fit values were not significant (Table 3).

Effect of nitrogen source on non-secretedand secreted chitinase activities

Non-secreted and secreted chitinase activities from Monilioph-

thora perniciosa were monitored using inorganic (NH4H2PO4)

and organic (yeast extract and peptone) nitrogen sources.

The inorganic source NH4H2PO4 used alone was insufficient

to allow the growth and survival of M. perniciosa, and there-

fore, no chitinase activity was observed in the corresponding

mycelium and culture medium (Table 2, assay 12). Among

the organic nitrogen sources and compared with the pep-

tone–yeast extract combination or yeast extract alone,

peptone enhanced non-secreted chitinase activity when com-

bined with chitin as carbon source (Fig 1A). However, chiti-

nase secretion in the culture medium greatly increased in

the presence of yeast extract alone, and significant responses

were also obtained when yeast extract was applied in combi-

nation with another nitrogen source (peptone or NH4H2PO4;

Fig 1B). This last result was obtained in culture medium for

cultures containing NAG (Fig 1B) as the carbon source.

Effect of carbon source on non-secretedand secreted chitinase activities

Non-secreted and secreted chitinase activities from Monilioph-

thora perniciosa were monitored using different carbon sour-

ces: chitin, glucose, and NAG. Non-secreted chitinase

activity was enhanced by chitin (peak production at 30 d cul-

tivation) and repressed by glucose regardless of the duration

of incubation. Non-secreted chitinase activity at 10 d occurred

in the presence of NAG with a higher yield (180 UA ng�1 pro-

tein h�2) than that obtained in the presence chitin (150 UA

ng�1 protein h�2; Fig 2A). Secretion of chitinase in the culture

medium was higher in the presence of NAG and lower in the

presence of chitin regardless of the duration of incubation,

even at the height of secretion, which occurred at 20 d. The

presence of glucose did not affect the recovery of chitinase

activity from the culture medium as strongly as it did in the

mycelium (Fig 2B).

Table 3 – Characteristics of the models used in the centralcomposite design

ModelF-value

R2 Lack-of-fitvalue

Chitinase activity Mycelium 3.41 0.771 71� 10�6

Culture medium 2.45 0.766 5� 10�6

Dry weight Mycelium 2.91 0.789 13� 10�3

Effect of incubation time on chitinase production

Non-secreted and secreted chitinase activities were analysed

after different incubation times, from 10 to 30 d. In the

mycelium, chitinase activity tended to increase with longer

incubation periods. Regarding the culture medium containing

peptone and chitin, peak activity was observed at 30 d (Fig 3A).

The highest chitinase secretion in the medium culture

occurred after 20 d incubation; longer or shorter incubation

periods were shown to be less effective (Fig 3B).

Effect of nitrogen source, carbon source,and incubation time on mycelium growth

The mycelium growth was quantified by weighing the dry my-

celium. The mycelium growth increased when yeast extract

and glucose were used as nitrogen and carbon sources,

respectively (Fig 4A); in these conditions, the highest growth

was obtained after 20 d incubation (Fig 4B,C). The lowest

growth was observed when the fungus was incubated with

chitin and NAG as carbon sources (Fig 4A,B), with NH4H2PO4

and peptone as nitrogen sources (Fig 4A and C), and at short

(10 d) and long (30 d) incubation times (Fig 4B,C). Moreover,

the mycelium cultivated in medium containing chitin (assay

10) was thinner and more dispersed than the mycelium culti-

vated on medium containing glucose, which was thicker and

more compacted (data not shown).

Discussion

Several prokaryotic and eukaryotic microorganisms are

potential producers of chitinolytic enzymes, and medium

composition can significantly affect their production (Nawani &

Kapadnis 2005; Souza et al. 2005). The use of statistical

methods based on experimental design, such as the response

surface methodology, enabled the evaluation of the simulta-

neous effects of culture medium components and of variables

(e.g. incubation time) on the enzymatic production. The use of

these methods enables a better understanding of the possible

interactions between evaluated variables (Nawani & Kapadnis

2005). The model developed here enabled the analysis of the

influence of both carbon and nitrogen sources and incubation

time on the chitinase activity of Moniliophthora perniciosa. The

regulation of the synthesis of chitinases is still little

understood (Souza et al. 2005), even for organisms considered

high chitinase producers, such as Trichoderma harzianum

(Nampoothiri et al. 2004). To our knowledge, this is the first

analysis of chitinase regulation in M. perniciosa and, more gen-

erally, in basidiomycetes.

This study focused on the activity of two chitinases: the

non-secreted and secreted. The intracellular localization of

chitinases has been previously demonstrated by Takaya

et al. (1998), and it seems that the greater portion of chitinases

remains in the cell wall where they could act on chitin (located

only in cell wall) and participate in cell wall modification dur-

ing cell growth and morphogenesis (Choquer et al. 2007). Some

of these chitinases may be covalently linked to the wall struc-

ture (Iranzo et al. 2002). Here, we assumed that non-secreted

Fig 1 – Surface plot of chitinase activity of Moniliophthora perniciosa as a function of nitrogen and carbon sources (in coded

values). (A) Mycelium. (B) Culture medium.

Chitinase regulation in Moniliophthora perniciosa 403

chitinases included intracellular chitinases (whose role and

localization remains to be resolved) and those tightly linked

to the cell wall. As suggested in other works, chitinases,

even from the same group, may be differentially regulated

(Choquer et al. 2007).

Chitin, glucose, and N-acetylglucosamine were used to

evaluate the effects of the carbon sources on non-secreted

and secreted chitinase activities. In cultures supplemented

by chitin, an increase in non-secreted chitinase activity was

observed at 30 d. However, at 10 d, non-secreted chitinase ac-

tivity was slightly higher with NAG (180 UA ng�1 protein h�2)

than with chitin (150 UA ng�1 protein h�2; Fig 2A). As chitin

is insoluble, the fungus is unable to utilize it without previous

hydrolysis into NAG or soluble oligomers. Although non-

secreted chitinase activity remained almost constant in the

presence of NAG throughout the induction time course, it sig-

nificantly increased in presence of chitin, suggesting that non-

secreted activity at 30 d may be induced by chitooligomers

and not by NAG. In support of this hypothesis, a plasma

membrane receptor has recently been discovered on plant

Fig 2 – Surface plot of chitinase activity of Moniliophthora pernicio

(A) Mycelium. (B) Culture medium.

cells, which recognizes chitin fragments for defence signalling

(Kaku et al. 2006; Choquer et al. 2007). Conversely, when the

medium was supplemented with glucose, non-secreted chiti-

nase activity was repressed whatever the duration of incuba-

tion (Fig 2A). However, glucose did not have a stronger

repressive action on the secreted chitinase activity (Fig 2B),

suggesting that these two categories of chitinases may be

differently regulated in the presence of glucose as the carbon

source. Similar results were obtained for other fungi, such as

Trichoderma harzianum (Nawani & Kapadnis 2005; Ulhoa &

Peberdy 1991) and Colletotrichum gloesporioides (Souza et al.

2005). However, the increase of the chitinase activity may

also be influenced by other carbon sources, such as NAG.

Barreto et al. (2004) demonstrated that NAG both up- and

down-regulated chitinase production in Metarhizium aniso-

pliae; when present in low concentrations (�0.05 %), NAG

induced the production and secretion of chitinase, but when

present in higher concentrations, it repressed chitinolytic

activity. In Aspergillus fumigatus (Escott et al. 1998) and Tricho-

derma harzianum (Nawani & Kapadnis 2005; Ulhoa & Peberdy

sa as a function of time and carbon source (in coded values).

Fig 3 – Surface plot of chitinase activity of Moniliophthora perniciosa as a function of nitrogen source and time (in coded

values). (A) Mycelium. (B) Culture medium.

404 M. A. Lopes et al.

1991), NAG repressed chitinase activity by negative feedback.

In M. perniciosa, NAG did not appear to exert an effect on

non-secreted chitinase activity (see discussion above); how-

ever, it did induce extracellular chitinolytic activity (Fig 2B).

Considering the relatively high concentration of NAG (0.5 %)

used in our experiments, the increase in activity of the

Fig 4 – Surface plot of Moniliophthora perniciosa mycelium dry w

a function of carbon source and time; (C) as a function of nitrog

secreted chitinase in M. perniciosa at 20 d was quite unex-

pected. However, because of the lack of information regarding

the regulation of chitinolytic activities in basidiomycetes, it

could be speculated that the regulation of the chitinase path-

way is very different from those fungi already described, espe-

cially when secreted and non-secreted chitinase activities are

eight: (A) as a function of carbon and nitrogen sources; (B) as

en source and time.

Chitinase regulation in Moniliophthora perniciosa 405

analysed separately. Alternatively, it could be that the thresh-

old of chitinase negative feedback is much higher in M. perni-

ciosa than in other fungi. The latter hypothesis may be

supported by the fact that non-secreted chitinase activity

remained almost constant throughout the induction time

course (Fig 2A), whereas secreted chitinase activity decreased

at 30 d, suggesting that chitinase secretion may submit to neg-

ative feedback by NAG at the end of the time course, (Fig 2B).

Complementary experiments testing chitinase regulation un-

der different concentrations of NAG may elucidate this and

enable better understanding of chitinase regulation.

Chitinase production in microorganisms is also influenced

by the nitrogen source. In Colletotrichum gloeosporioides, organic

sources of nitrogen, such as yeast extract, peptone, urea, and

glutamic acid, resulted in better chitinase production than

the inorganic sources (Souza et al. 2005). In M. perniciosa,

a greater increase of the mycelium chitinolytic activity was

observed when peptone was used, mainly when the medium

culture also contained chitin (Fig 3A). Conversely, the extracel-

lular chitinase activity was increased by the yeast extract when

combined with NAG (Fig 3B). The effect of interactions between

carbon and nitrogen sources on chitinase activity were

observed in T. harzanium, which showed a relatively high in-

crease of the extracellular chitinolytic activity in culture media

containing yeast extract and chitin (Nampoothiri et al. 2004).

The effect of the incubation time must be also considered;

when M. perniciosa was cultivated on chitin or peptone, its my-

celial chitinase activity was at its highest after 30 d, whereas

the extracellular chitinase activity was at its peak after 20 d.

In all the experiments performed in the present study, the

chitinase activity was higher in the mycelium than in the cul-

ture medium (Table 2). The difference between non-secreted

and secreted chitinase activities varied from 1.8 to 115 times,

for assays 15 and 8, respectively. Only assay 9 presented the

same chitinase activity in both mycelium and extracellular

extracts. On average the mycelium chitinase activity was 17

times greater than the extracellular chitinase activity. These

results indicated that M. perniciosa, under the studied condi-

tions, did not have a high chitinase secretion activity. If

chitinases were produced but not secreted, their role might

be related to fungal development, specifically to the mycelium

growth. As showed in the Fig 4, mycelium growth was directly

influenced by the substrate, and this may be correlated to

non-secreted chitinase activity. The highest mycelium growth

was observed when non-secreted chitinase activity was at its

lowest, i.e. when the fungus was grown on glucose and yeast

extract as sources of carbon and nitrogen, respectively.

More studies are underway to investigate the factors influ-

encing the induction–repression system of chitinases from

M. perniciosa, to explore the possibility of targeting them as

methods of disease control for witches’ broom. The sample

of fungal specimen was deposited in the Collection of Microor-

ganism Culture of Bahia (CCMB), Feira de Santana, BA, Brazil.

Acknowledgement

This research was supported by the Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq) and

Programa de Apoio ao Desenvolvimento Cientıfico e Tecnolo-

gico (PADCT). The Research Support Foundation of the State of

Bahia (FAPESB) funded M.A.L. and the PROIIC program of the

Universidade Estadual de Santa Cruz funded D.S.G. We thank

Alan Pomela for kindly providing Moniliophthora perniciosa,

Karina Gramacho for helpful advices and Marcio Costa for

critical reading of the manuscript.

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