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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
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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).
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