Article Citation: Berecochea-López A, Ragazzo-Sánchez JA, Allende-Molar R, Avila-Quezada GD and Calderon-Santoyo M. Colletotrichum gloeosporioides from Mango Ataulfo: morphological, physiological, genetic and pathogenic aspects. Journal of Research in Biology (2015) 5(2): 1641-1647

Jou

rn

al of R

esearch

in

Biology

Colletotrichum gloeosporioides from mango Ataulfo: morphological,

physiological, genetic and pathogenic aspects

Keywords: C. gloeosporioides, C. acutatum, Pectate Lyase

ABSTRACT: Colletotrichum causes anthracnose in crops around the world producing postharvest losses up to 60%. There are a great variety of Colletotrichum strains isolated from mango orchards. Thus, it is important to characterize their pathogenicity, as well as to perform a correct identification, in order to implement good strategies to eradicate the produced disease. The aim of this work is to identify Colletotrichum spp. and to determine the production of Pectate Lyase (PL) as a virulence factor in the pathogenicity process. Macroscopic characteristics of isolated colony vary from grey to salmon, sometimes showing luxuriant orange conidial masses with grey or white bottom. Conidia vary from 10.39 to 14.83 × 2.75 to 3.40 μm corresponding to C. gloeosporioides or C. acutatum according to Sutton. Growth rates vary from 0.1948 to 0.2239 day-1. The pectate lyase activity was induced by mango cells (240.81 VS 398U/L). According to CgInt and ITS4 PCR amplification M2V and SA correspond to C. gloeosporioides.

1641-1647 | JRB | 2015 | Vol 5 | No 2

This article is governed by the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which gives permission for unrestricted use, non-commercial, distribution and reproduction in all medium, provided the original work is properly cited.

www.jresearchbiology.com Journal of Research in Biology

An International

Scientific Research Journal

Authors:

Berecochea-López A1,

Ragazzo-Sánchez JA1,

Allende-Molar R2,

Avila-Quezada GD3 and

Calderon-Santoyo M1.

Institution:

1.Laboratorio Integral de

Investigación en Alimentos,

Instituto Tecnológico de

Tepic.

2.CIAD-Unidad Culiacán

Carretera El Dorado Km5.5,

Col. Campo El Diez,

Culiacán Sinaloa 80129

Mexico

3.CIAD-Unidad Delicias Av

4ta Sur 3820, Fracc.

Vencedores del Desierto .

Delicias, Chihuahua 33089

Mexico

Corresponding author:

Calderon-Santoyo M

Web Address: http://jresearchbiology.com/

documents/RA0388.pdf

Dates: Received: 18 Oct 2013 Accepted: 26 Oct 2013 Published: 18 Mar 2015

Journal of Research in Biology

An International Scientific Research Journal

ORIGINAL RESEARCH

ISSN No: Print: 2231 –6280; Online: 2231- 6299

INTRODUCTION

Mango has short time storage, principally due to

the susceptibility of attack by the phytopathogens.

The principal mango disease is anthracnose caused by

Glomerella cingulata (Stoneman) Spauld. and

H. Schrenk (anamorph: Colletotrichum gloeosporioides

(Penz.) Penz & Sacc.) In Penz., C. gloeosporioides

(Penz). var. minor. J.H. Simmonds (Collmer et al., 1988)

and C. acutatum J.H. Simmonds. Losses in farming and

postharvest caused by anthracnose are estimated between

15 and 50% (Freeman et al., 1998).

The production of hydrolytic enzymes have been

established among the mechanisms of pathogenicity in

fungi pathogenic to fruit. In consequence, the analysis of

enzyme production during the pathogenicity process

provides important information concerning the

mechanisms used by the fungus to penetrate and infect

the fruit (Prusky et al., 2001).

The Pectate Lyase (PL) is a pectinase involved in

the hydrolysis of the cell wall by Colletotrichum spp.,

specifically in the tissue maceration. This mechanism has

been elucidated in the avocado infection process, but

there are no reports concerning the mango fruit (Yakoby

et al., 2000).

There is diversity of strains of Colletotrichum

spp. that could be involved in the anthracnose disease

development in mango fruit. Differentiating between

Colletotrichum species responsible for anthracnose

disease is important for understanding the epidemiology

of this disease and developing better control strategies of

postharvest diseases (Fitzell and Peak, 1984). Then it is

important to realize an adequate identification and to

characterize physiologically the Colletotrichum species

(Schaad and Frederick, 2002; Sanders and Korsten,

2003).

The aim of this work is to characterize

Colletotrichum spp. and to determine the production of

Pectate Lyase (PL) as a virulence factor in the

pathogenicity process.

MATERIALS AND METHODS

Fungal isolates

Mango fruits cv. “Ataulfo” of different sizes and

displaying symptoms of anthracnose as well as

inflorescences were collected to identify the pathogens

involved in this disease. They were obtained from four

orchards viz: Aticama, Miramar, Mpio. San Blas and

Nayarit.

Colletotrichum spp. were isolated within 18–24 h

after harvesting fruits with symptoms, by excising a

small piece of necrotic pericarp with healthy tissue

margins. The fruits had previously been disinfected in

1% sodium hypochlorite for 3 min and rinsed three times

with sterile distilled water. Tissue samples were placed

on petri plates containing Potato Dextrose Agar (PDA)

and incubated at room temperature (25°C) for seven days

until mycelia development. Then, monoconidial cultures

were inoculated into Water Agar (WA). One single

germinated conidia, was transferred with a sterile needle

to PDA medium amended with 10% tartaric acid at

14 mL L-1. All single-conidia isolates obtained were used

for further characterization and pathogenicity test. In a

number of cases, more than one isolate was obtained

from each sample (Table 1).

Colletotrichum reference strains were obtained

from CIAD Unity Cuauhtemoc (Strain DG) and from

CIAD Unity Delicias (Strain GAQ37).

Morphological and growth characteristics

The description of Mordue (Mordue, 1971;

Freeman et al., 1998) was used as a reference to identify

the fungi at the genus level, and C. acutatum and

C. gloeosporioides were identified according to Sutton’s

key (Sutton, 1992). Small portions of (approximately

5 mm) two plugs of agar containing mycelia from each

culture of Colletotrichum spp. were transferred onto Petri

plates containing PDA to study morphological

characteristics. The plates were incubated at about 24°C

with a 12 h light - dark cycle for seven days. The colony

diameters (mm) were recorded every day for one week to

López et al., 2015

1642 Journal of Research in Biology (2015) 5(2): 1641-1647

determine the growth rates. The length and width of at

least 50 conidia were measured, and the colony colour

was recorded for each isolate. The size, shape and colour

of the conidial masses and other key characteristics of

each structure were also scored.

Genomic DNA extraction

Each isolate was cultured on PDA at room

temperature for 10 days. After this time, the mycelium

was scraped from the surface of the plate and crushed

with a mortar in 1 mL of lysis solution (2% Triton-X

100, 1% SDS, 100 mmol L -1 NaCl, 10 mmol L -1

Tris-HCl, pH 8.0) containing 10 µL of proteinase K (10

mg mL-1). Subsequently, 600 µL of the sample were

transferred to a 2.0 mL eppendorf tube and the DNA

pellet of each tube was suspended in 50 µL of TE buffer

(10 mmol L-1 Tris-HCl, pH 8, 1 mmol L-1 EDTA).

The quality of the DNA was verified by electrophoresis

in a 1.0% agarose gel in 1X TAE buffer (Tris Acetate-

EDTA) run at 87 Vcm-1 for 1 h. The gel was stained with

gel RED, and the bands were visualized under a Gel Doc

2000 UV transilluminator (Bio-Rad). The DNA

concentration was quantified using a Nanodrop

spectrophotometer (Thermo Scientific), and the samples

were diluted to 20 ng µL-1 for PCR reactions.

PCR amplification of ribosomal RNA genes

T h e p r i m e r s C g I n t ( 5 ’ -

GGCCTCCCGCCTCCGGGCGG-3’) and ITS4

(5’- TCCTCCGCTTATTGATATGC- 3’) were used to

amplify an approximate fragment of 500 bp. A PCR

master mix was prepared containing 0.2 µL of Taq DNA

polymerase buffer (5 u/µL), 1.5 µL MgCl2 (25mM), 0.5

µL of dNTP (10mM), 10 ng of genomic DNA, 0.5 µL of

each primer, 5 µL of PCR buffer 10´ and nanopure

sterile water to a final volume of 25 µL. PCR

amplifications were performed with an initial

denaturation at 94°C for 2 min; 30 cycles of denaturation

at 94°C for 1 min, annealing at 45°C for 2 min, and

extension at 72°C for 3 min; followed by a final

extension at 72°C for 5 min (Mills et al., 1992; Tapia

et al., 2008). All PCR reactions were carried out in a

Peltier Thermal Cycler C1000 (Bio-Rad), and the PCR

products were verified by loading of 5 µL in a 1.5%

agarose electrophoresis gel, which was stained as

described above.

Sensitivity to benomyl

PDA petri plates were simultaneously inoculated

with a conidial suspension of each isolate as described

above in order to determine the sensitivity of isolates to

benomyl (Freeman et al., 1998). Filter paper discs (1 cm

in diameter), previously saturated with 100 μl of 0, 300,

600, or 1200 μg/ml of benomyl and air-dried, were

placed on the surface of inoculated PDA plates. The

plates were incubated for 3 days at 25°C, the radius of

the inhibition zone was recorded for each fungicide

concentration evaluated, and plates were photographed.

Three replicates of each isolate for each fungicide

concentration were evaluated, and the experiment was

performed twice. The factorial experiment was evaluated

using general linear model procedures and means were

separated using Tukey’s studentized range test provided

in the statistical algorithms of SAS version 6.04

(SAS Institute) (Freeman et al., 1998).

Pectate lyase activity

Pectate Lyase (PL) was assayed as described by

(Collmer et al., 1988). PL activity was conducted with

5 μg of protein, which was on the linear portion of the

curve for 5 min in a spectrophotometer (UV-vis 6405,

Janway) at 232 nm. PL activity was calculated using the

increase in absorbance caused by the accumulation of the

4,5- unsaturated galacturonide product of

polygalacturonic acid (Fluka) for 10 minutes, at 37ºC.

The molar extinction coefficient for the unsaturated

product at 232 nm is 4,600 per M per cm. One unit of PL

was considered as the necessary activity for producing

1 μmol of 4,5-unsaturated products for 10 min under the

conditions of the assay.

The substrate stock solution was prepared by

adding Tris HCl 50mM, pH 8.5, CaCl2 and deionized

López et al., 2015

1643 Journal of Research in Biology (2015) 5(2):1641-1647

water and 0.2% polygalacturonic acid. Reaction mixtures

consisted of 950 µl of substrate solution and 50 μl of

protein extract concentrated media, followed by

incubation for 10 min at 37ºC. The absorbance was read

at 235nm.

RESULTS AND DISCUSSION

Fungal isolates, morphological and growth

characteristics

Six isolates showing macroscopic characteristics

vary from grey to salmon, sometimes showing luxuriant

orange conidial masses with grey or white bottom were

obtained. Sutton (1992) appointed some characteristics

for C. acutatum as white mycelia becoming gray to

grayish brown, on contrary to Zulfiqar et al. (1996) who

described initial white mycelia for C. acutatum and

posterior emergence of masses of pink or orange conidia.

Then, it is difficult to establish criteria for the

identification of Colletotrichum species based on

morphological characteristics. Sutton, (1992) concluded

that the size varies for C. gloeosporioides conidia from

12-17 x 3.5 -6 mm and for C. acutatum from 8.5-16.5 x

2.5- 4 mm. In this sense, the isolates M2V, SA, GAQ37,

F1 and A71NB correspond to C. gloeosporioides (13.4–

24 x 4.0–5.9) and the isolate DG correspond to C.

acutatum (11.8–19.1 x 3.2–8.5) (Figure 1 and Table 1).

Growth rates

C. gloeosporioides generally present higher

amounts of growth rates than C. acutatum. According to

this, the isolates M2V and DG probably correspond to C.

López et al., 2015

1644 Journal of Research in Biology (2015) 5(2): 1641-1647

Figure 1. Microscopic characteristics of Colletotrichum.

Spat 40´ (M2V, SA, A71NB and F1 are isolates. DG and

GAQ37 are reference strains).

Table 1. Conidia measures of Colletotrichum spp. (M2V,

SA, A71NB and F1 are isolates. DG and GAQ37 are

reference strains)

Isolates Length Width

M2V

SA

F1

A71 NB

10.39

12.42

11.00

11.24

2.75

2.77

3.34

3.21

DG

GAQ37

16.14

9.78

4.02

3.40

Figure 2. Agarose gel showing the DNA fragment ampli-

fied from the oligonucleotides CgInt and ITS4 for the

Colletotrichum gloeosporioides. M: Molecular weight

marker 100 pb, 1: F1, 2: M2V, 3: SA, 4: DG, 5: GAQ37,

6: A71 B, 7: Positive control Colletotrichum gloeospori-

oides (Yucatán).

Isolates

Mean growth rate (mm Day-1)

M2V SA F1

A71NB

0.1948 0.2239 0.2047 0.1990

DG GAQ37

0.1871 0.2109

Table 2. Growth rates of Colletotrichum spp. isolates

acutatum and SA, F1 and GAQ37 to C. gloeosporioides

(Table 2), but this classification is not definitive.

Molecular identification

According to the use of the primers CgInt and

ITS4 specific for C. gloeosporioides, an amplification

product nearer to 500 bp was obtained. This fragment

matches the sizes of DNA for C. gloeosporioides

(Adaskaveg and Hartin, 1997). Thus, the isolates M2V

and SA were positive to the primers used as well as the

reference strains DG and GAQ37 (Figure. 2). The

isolates F1 and A71NB were not identified as C.

gloeosporioides (Figure. 2), then, these are probably

C .

acutatum.

Sensitivity to benomyl

Colletotrichum strains were classified as C.

gloeosporioides and C. acutatum according to their

sensibility to benomyl. The isolate DG was sensitive to

benomyl and classified as C. gloeosporioides. The

isolates M2V, SA, GAQ37, F1 and A71 NB did not

show sensibility to benomyl and they were classified as

C. acutatum (Table 3).

Pectate Lyase activity

Higher amount of PL activity was observed in

the isolates cultured in M3S liquid medium with

presence of mango cells when compared to those

cultured in the absence of mango cells. The isolates

M2V, SA and F1 showed a difference between absence

and presence of mango cells. It is also inferred by a

mechanism of PL induction by the components of mango

cells (p<0.05) (Table 4).

The electroblotting of polyacrylamide gels to

14% (Figure 3) shows that the bands are observed in

more intense blue color with the presence of the

inductor. The molecular weight marker is located in lane

1. The pectate lyase is obtained at 40kD weights so that

López et al., 2015

1645 Journal of Research in Biology (2015) 5(2): 1641-1647

Table 3. Sensitivity test to benomyl of isolates and reference strains inoculated at 25

± 2 ° C for 6 days

Isolates 0.0 g/L Benomyl 0.3 g/L Benomyl 0.6 g/L

Benomyl

1.2 g/L

Benomyl

M2V 1 0 0 0

SA 1 0 0 0

F1 1 0 0 0

A71NB 1 1 1 0

DG 1 0 0 0

GAQ37 1 0 0 0

40kD

Figure 3. Western blot of extracts from Colletotrichum

spp. isolates culture with and without mango cells.

Table 4. Enzymatic activity of Pectate Lyase obtained from Colletotrichum spp.

isolates in presence and absence of mango cells cv “Ataulfo” as inductor.

Isolates Without inductor (U/L) With mango cells as inductor (U/L)

M2V 234.7a 412.1b

SA 238.0a 410.96b

F1 102.7a 271.6b

DG 387.69a 416.13a

Different letters show significant differences by LSD with p<0.05.

the bands were between carbonic anhydrase (45.7kD)

and soybean trypsin inhibitor (32.5kD).

CONCLUSION

The morphological and physiological

characteristics of Colletotrichum isolates are not

definitive to classify them up to the species

gloeosporioides or acutatum, but the molecular criteria

ie., Pectate lyase enzyme is involved in the process of

pathogenicity of Colletotrichum. sp.cv “Ataulfo”

contributes for their better identification. However, other

enzymes may also be involved. The identification and

characterization of Colletotrichum spp. responsible for

anthracnose in Nayarit will contribute to design better

monitoring methods in pre- and postharvest strategies.

ACKNOWLEDGEMENT

The authors thank COCYTEN (Nayarit, Mexico)

for the scholarship granted to Arlet BERECOCHEA

LÓPEZ and CONACYT (Mexico) for their support in

conducting the work throughout this project (code

SEP-CONACYT 26075).

REFERENCES

Adaskaveg JE and Hartin RJ. 1997. Characterization

of Colletotrichum acutatum isolates causing anthracnose

of almond and peach in California, Phytopathology. 87

(9): 979-987.

Collmer A, Ried JL and Mount MS. 1988. Assay

methods for pectic enzymes, Methods in Enzymology.

161: 329-335.

Fitzell RD and Peak CM. 1984. The epidemiology of

anthracnose disease of mango: inoculum sources, spore

production and dispersal, Annals of Applied Biology.

104(1): 53-59.

Freeman S, Katan T and Shabi E. 1998.

Characterization of Colletotrichum species responsible

for anthracnose diseases of various fruits, Plant Disease.

82(6): 596-605.

Mills PR, Brown AE and Sreenivasaprasad S. 1992.

Detection and differentiation of Colletotrichum

gloeosporioides isolates using PCR, FEMS

Microbiology Letters. 98(1-3): 137-143.

Mordue JEM. 1971. Glomerella cingulata. CMI

Descriptions of Pathogenic Fungi and Bacteria No. 315.

Commonwealth Mycological Institute, Kew, England.

2p.

Prusky D, McEvoy JL, Leverentz B and Conway WS.

2001. Local modulation of host pH by Colletotrichum

species as a mechanism to increase virulence, Molecular

Plant-Microbe Interactions. 14(9): 1105-1113.

Sanders GM and Korsten L. 2003. A comparative

morphological study of South African avocado and

mango isolates of Colletotrichum gloeosporioides,

Canadian Journal of Botany. 81(8): 877–885.

Schaad NW and Frederick RD. 2002. Real-time PCR

and its application for rapid plant disease

diagnostics. Canadian Journal of Plant Pathology. 24(3):

250-258.

Sutton BC. 1992. The genus Glomerella and its

anamorph Colletotrichum. In: Bailey JA, Jeger MJ, eds.

Colletotrichum: Biology, Pathology and Control.

Wallingford, UK: CAB International. 1–26.

Tapia-Tussell R, Quijano-Ramayo A, Cortes-

Velazquez A, Lappe P, Larque-Saavedra A and Perez

-Brito D. 2008. PCR-based detection and

characterization of the fungal pathogens Colletotrichum

gloeosporioides and Colletotrichum capsici causing

anthracnose in papaya (Carica papaya L.) in the Yucatan

península, Molecular Biotechnology. 40(3): 293-298.

López et al., 2015

1646 Journal of Research in Biology (2015) 5(2): 1641-1647

Yakoby N, Kobiler I, Dinoor A and Prusky D. 2000.

pH regulation of Pectate Lyase secretion modulates the

attack of Colletotrichum gloeosporioides on avocado

fruits, Applied and Environmental Microbiology.

66(3):1026-1030.

López et al., 2015

1647 Journal of Research in Biology (2015) 5(2): 1641-1647

Submit your articles online at www.jresearchbiology.com

Advantages

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing You retain your copyright

submit@jresearchbiology.com

www.jresearchbiology.com/Submit.php