Morphological And Molecular Identification of Phaeoacremonium ...

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Morphological And Molecular Identification of Phaeoacremonium ...

Journal of Basrah Researches ((Sciences)) Volume 37. Number 4. E ((2011))

Available online at: http://www.basra-science-journal.org

ISSN18172695

Morphological And Molecular Identification of Phaeoacremonium

aleophilum Associated with Grapevines Decline Phenomenon in Duhok

Governorate

1 Raed A. Haleem , 2 Samir K. Abdullah and 3 Jaladat M.S. Jubraeel

1 Department of Plant Protection, College of Agriculture, University of Duhok, Iraq

2 Department of Biology, College of Science, Zakho University, Iraq , samer_abdalh@yahoo.com

3 Scientific Research Centre, University of Duhok, Iraq

Abstract

Decline symptoms on grapevine included plants that failed to thrive normal with reducing

shoot growth and chloratic interveinal areas that latter became necrotic. In a cross section of

grapevine arms, the internal wood tissue were frequently dark brown to black with a wedgeshaped

necrotic sectors. Phaeoacremonium aleophilum was isolated from infected tissues of

declined plants in pure culture and identified on the basis of its morphological and cultural

characteristics. For accurate identification of P. aleophilum the PCR technique was employed.

Ten isolates were selected from different locations. These isolates were subjected to specific

PCR assay. The specific primers for P. aleophilum were used to amplify the ITS region of

nuclear ribosomal DNA (rDNA) containing ITS1, ITS2 and the intervening 5.8 rRNA genes.

PCR results obtained from Phaeoacremonium isolates indicated that only three isolates were

related to P. aleophilum. The remaining isolates may represent different species of

Phaeoacremonium. P. aleophilum is reported for the first time in Iraq.

Keywords: grapevines decline, Phaeoacremnium aleophilum, molecular detection.

Introduction

The genus Phaeoacremonium is

intermediate between Acremonium Link,

Fr. and Phialophora Medlar.

Phaeoacremonium parasiticum, under its

original name Phialophora parasitica

Ajello, Georg & C.J.K. Wang is the type

species for the genus. Phaeoacremonium

can be distinguished from Phialophora by

its aculeate phialides and inconspicuous,

non-flaring collarettes, and from

Acremonium by its pigmented vegetative

hyphae (Crous et al., 1996). Togninia minima

(Diaporthales: Ascomycota) was recently

confirmed as the sexual stage of

1

Phaeoacremonium aleophilum. T. minima is

characterized by having dark globose longbeaked

and non-stromatic perithecia

(Rooney-Latham et al., 2005a,b).

Phaeoacremonium species have wide

host range and world wide distribution ,

however, majority of them are found on

Vitis vinifera, others were reported from

Olea europaea, Fraxinus spp., Prunus spp.,

Salix spp., and Quercus spp. Few species are

parasitic on human (Auger et

al.2005,Eskalen et al.2005, Mostert et al.

2005b).


Haleem, Abdullah & Jubraeel: Morphological And Molecular Identification of Phaeoacremonium aleophilum…

Six species of Phaeoacremonium were

originally identified based on morphology

and cultural characters (Crous et al., 1996).

It soon became apparent that the taxon once

referred to as ‘Cephalosporium’ species or

P. Chlamydosporum represented a new

genus, Phaeomoniella Crous & W. Gams,

which resided within the Chaetothyriales

(Crous and Gams, 2000). Morphological

characters that were useful in distinguishing

species included conidiophore morphology,

phialide type and morphology, the size of

hyphal warts, and to a lesser extent conidial

size and shape; cultural characters that were

useful included colony colour on 2% malt

extract agar (MEA), yellow pigment

production on potato-dextrose agar, growth

rate at 25°C and maximum growth

temperature (Mostert et al., 2005b). Yellow

pigment production on oatmeal agar was

used by Dupont et al. (2000). The genus

Phaeoacremonium is characterized by its

mycelial bundles, branched or simple

conidiophores, slender phialides occurring

in three size classes, narrowly funnel-shaped

collarettes at the apex of the phialides,

conidia aggregated into slimy heads and

conidial shape ranging from mostly oblongellipsoidal

to allantoid. Generic descriptions

of Phaeoacremonium have been published

by Crous et al (1996) and Mostert (2005b).

Molecular characters have played an

important role in the detection and

Materials and Methods.

Fungal isolation.

Isolation was done in two methods:

1- Isolation from complete vine

tissues.

Complete vine tissues were sampled,

from cane (bark and wood), bud, trunk or

arm (bark and wood), leaves, clusters and

roots. Small pieces of tissue from the

margin between necrotic and apparently

healthy tissue were surface sterilized by

placing in 70% ethanol for 30 s, 1% NaOCl

for 1 min and again in 70% ethanol for 30 s

and then dried by filter papers. Sterilized

tissues pieces were plated onto 2% potato

dextrose agar (PDA) (Himedia Laboratories

identification of Phaeoacremonium species

(Tegli et al., 2000; Dupont et al., 2002;

Mostert, 2006; and Aroca et al., 2008).

Species-specific primers based on the tubulin

and actin genes have been developed

(Mostert, 2006) and can be used in

multiplex polymerase chain reactions (PCR)

for the identification of unknown isolates.

To date 22 species of Phaeoacremonium

have been isolated from grapevines (Crous

et al., 1996; Mostert et al., 2005b; Mostert,

2006; Essakhi et al., 2008; Gramaje et al.,

2009). Sixteen species of Phaeoacremonium

were described based on molecular

characters, the internal transcribed spacer

(ITS) regions 1 and 2, the 5.8S rDNA

(Dupont et al., 2000) and the -tubulin gene

(Groenewald et al., 2001). Subsequent

studies included the actin and calmodulin

gene regions (Mostert et al., 2005b; and

Mostert, 2006) of the species occurring on

grapevines. P. aleophilum is the most

common and widely distributed species

(Crous et al.,1996, Larignon and Dubos

1997; and Mugnai et al., 1999) and P.

parasiticum is encountered frequently

(Dupont et al., 2002; and Mostert, 2006).

The objective of this study is isolation

and identification of Phaeoacremonium

species associated with declined grapevine

trees in Duhok governorate, Kurdistan

region of Iraq, based on morphological and

molecular techniques.

Pvt. Ltd. - India) containing 0.25 mg/ml

chloramphenicol. Hyphae growing out from

the tissue pieces were cut and subcultured

onto fresh PDA plates, and incubated at

25±2 °C (Van Niekerk et al, 2004).

Sporulation was enhanced by culturing the

isolates on 2% water agar bearing pieces of

autoclaved grapevine canes at 25 °C with a

12/12 h photoperiod (Luque et al., 2005).

2- Moist chamber method.

Cuttings were made from various

grapevine parts including canes, arms and

trunk. All segments were placed in 90 mm

petridishes containing sterilized moist filter

paper. Plates were incubated at room

2


Journal of Basrah Researches ((Sciences)) Volume 37. Number 4. E ((2011))

temperature until fungal growth observed.

Propagules (spores, mycelia) were

transferred to Potato-dextrose-Agar (PDA)

plates. Pure cultures of each isolate were

obtained by excising a hyphal tip from

colony margins and plating it onto fresh

PDA.

Phenotypical characterization

All isolates were grown on PDA and

MEA at 25°C in darkness or under NUV +

fluorescent illumination with a 12-h

photoperiod (Philips /36W) for 10 - 15 days

until cultures sporulated. Isolated strains

were identified based on the characters in

culture and on natural substrates (Crous et

al, 1996; Mostert et al .2005a,b).

DNA extraction and PCR

amplification of Phaeoacremonium

aleophilum.

- Fungal Isolates

Ten isolates were selected to confirm the

identification by a specific primer of the ITS

region. Isolates were collected from five

locations in Duhok governorate., as show in

table (1).

Table (1): Isolates of Phaeoacremonium spp. from different grape vineyards of Dohuk governorate.

Phaeoacremonium spp.

Isolates

DP1

DP2

DP3

DP4

DP5

DP6

DP7

DP8

DP9

DP10

- Total genomic DNA extraction.

Genomic DNA was extracted according to a

method reported by Borges et al (1990).

Fungi were grown in 2% malt extract broth

with gentle shaking. Freezing fungal

mycelium in liquid nitrogen and grinding

the frozen sample to a powder by means of a

pestle and mortar. Powdered mycelium

(0.5g) was transferred to a sovrall tube

containing 5 ml of cold SDS buffer and

thoroughly shaken for 15 minutes. The

mixture was then immediately extracted

with 1 vol. distilled saturated phenol by

shaking several time for 10 minutes and

centrifuged at 4000 rpm for 15 minutes.

After a phenol extraction, the aqueous phase

was extracted with 1 vol. chloroform

isoamyl by shaking for 15 minutes before

centrifugation for 30 minutes. Ten percent

of the vol. of 7.5M ammonium acetate was

added to the resulting aqueous phase. DNA

was then precipitated by addition of 2 vols.

Isolated part Cultivar Geographical

location

Arm's wood Rashmew Nizarke

Cane's wood Kamali Malata nursery

Root

Kamali Badi

Cane's wood Taefi Zawita

Root

Rashmew Nizarke

Arm's wood Kamali Bajelor

Cane's bark Kamali Badi

Arm's wood Taifi

Bajelor

Arm's wood Taifi

Bajelor

Root

Rashmew Berebahar

3

of cold absolute ethanol, before incubation

overnight at -20 °C ,and recovered either by

spooling it out or by centrifugation at 4000

rpm for 20 minutes. The supernatant was

discarded and the pellet was washed with

1ml of 70% cold ethanol, incubated

overnight at -20°C and then centrifuged at

4000 rpm for 10 minutes. The supernant was

discarded and the DNA pellet was then airdried

at room temperature for 30

minutes.The DNA pellet was redissolved in

300 µl TE buffer and stored at -20°C until

use.

- PCR amplification of ITS region.

The specific primers of

Phaeoacremonium aleophilum were used to

amplify the ITS region of nuclear ribosomal

DNA (rDNA), containing ITS1, ITS2 and

the intervening 5.8 rRNA gene (Pal1F 5’-

AGGTCGGGGGCCAAC-3’, Pal2R 5’-

AGGTGTAAACTACTGCGC-3’) (Tegli et


Haleem, Abdullah & Jubraeel: Morphological And Molecular Identification of Phaeoacremonium aleophilum…

al, 2000). The PCR reactions were carried

out in a total volume of 25 µl, in thinwalled,

0.5 µl Eppendorf tubes.Master mix

was prepared for 12 samples of each fungus

(10 isolates plus 2 control) by mixing 30 µl

of 10XPCR, 30 µl of dNTPs, 24 µl forward

primer, 24 µl Reverse primer, 12 µl MgCL2,

4.8 µl of Taq polymerase enzyme and deionized

distilled water was added to a final

volume of 252µl. The solution mixed and

spun for 10 second in a microcentrifuge.

Later, the mixture was dispensed in PCR

tubes. All these steps were done on ice.

Amplification was carried out in an

automated thermal cycler (Delphy 1000,

Oracle Biosystems, MJ Research Inc.,

Results and Discussion

Phenotypical characterization

Phaeoacremonium aleophilum W. Gams,

Crous, M. J. Wingf. et L. Mugnai.

Mycologia 88:791 (1996). Fig. (1) A – I.

Telemorph: Togninia minima (Tul.and C.

Tul.) Berl., Icon. Fung. (Abellini) 3:9

(1900).

Cultural characters: colonies on MEA,

reached a diameter of 22 mm after 20 days

of incubation at 25°C. Flat, mostly felty

texture with entire edge, Pale yellow color

in above and in reverse. Colonies on PDA,

reached a radial of 25.5 mm after 20 days.

Flat, wooly texture with entire edge, Dark

blond to brownish grey towards the edge

above, in reverse pale brown to dark brown

towards the edge.

Aerial structure: Hyphae are verruculose,

medium to pale brown, and 1.5 - 2.5 µm

wide. Conidiophores are mostly short and

usually unbranched, 0-3 septate. 17 - 29 µm

long and 2 - 2.5 µm wide. The apical cell of

conidiophores usually produces one

phialide. Phialides terminal or lateral,

mostly monophialidic, smooth to

verreculose, subhyline; type II and type III

phialides are most common. Type I

phialides are cylindrical occasionally wider

at the base, 4 - 9 × 1 – 1.5 µm (av. 5 × 1.5)

µm. Type II phialides are either elongateampulliform

and attenuated at the base or

are navicular, tapering towards the apex, 10-

4

Watertown, MA, USA) according to the

following programs: An initial denaturation

at 95°C for 3 min, after which 30

cycles of de-naturation (2 min at 95°C),

primer annealing (25 sec at 64°C) and

primer extension (2 min at 72°C) were

performed. A final extension was performed

at 72°C for 10 min. Amplification reactions

were conducted at least twice, in two

separate experiments. For each isolate, 5 µl

of PCR products were mixed with 7µl

loading buffer and then analyzed by

electrophoresis in 2% (w:v) agarose gels

with 1xTBE buffer visualized by UV

fluorescence.

14 × 1.5-2.5 µm (av. 11 × 2) µm. Type III

phialides are subcylindrical or elongate -

ampulliform and attenuated at the base, 15 -

20 × 1.5 - 2 µm (av. 18 × 2) µm. Conidia

are mostly oblong-ellipsoidal or cylindrical,

2.5 - 6 ×1 - 2 µm (av. 3.5 × 1.5) µm. This

description was in agreement with the other

investigations (Crous et al., 1996; Mostert et

al., 2005b; and Mostert, 2006).

Molecular detection of P.

aleophilum

- Genomic DNA isolation and

purification.

Suitable yields of genomic DNA were

obtained from repeated experiments with an

average yield of 1.5-6.70 µg/ml and a purity

of about (1.6-1.8) determined by

spectrophotometer ratio A 260/A 280. The

molecular weight of DNA samples was

estimated using 1% agarose gel

electrophoresis containing DNA sample as

control (Fig. 2,). Ratios above 2.0

correspond to RNA contamination, while

ratios below 1.6 suggest protein

contamination (Sinha et al., 2001).

- Species specific primers of P.

aleophilum isolates.

On the basis of the sequence data of the

ITS regions, the primer pairs Pal1-Pal2 were

designed by Tegli et al. (2000) to amplify

specific DNA fragments using genomic


Journal of Basrah Researches ((Sciences)) Volume 37. Number 4. E ((2011))

DNA from P. aleophilum (Pal) isolates.

Two primer pair Pal1F-Pal2R specifically

amplified a fragment of about 400 bp in

three Pal tested isolates (DP1, DP2, and

DP3), as shown in Fig. (3), but no

amplification was detected when other

isolates were tested.

Based on morphological

characteristics, these ten isolates were very

close to each other, thus they were identified

as P. aleophilum. However, the molecular

detection confirmed that only three of them

were related to P. aleophilum (DP1, DP2,

and DP3). Thus the sequencer needed to

obtain data of the ITS region to identify

remaining species. To date, 22 species of

Phaeoacremonium have been isolated from

5

grapevines (Crous et al., 1996; Mostert et

al., 2005b, 2006; Essakhi et al., 2008; and

Gramaje et al., 2009). It was observed from

previous studies that there were only six

species of Phaeoacremonium spp. described

depending on the morphological characters,

whereas the other species were identified

depending on the molecular analysis

(Mostert et al., 2006; Essakhi et al., 2008;

and Gramaje et al., 2009). In any case, it

seems that the identification of

Phaeoacremonium species by their

morphological and biological characteristics

should be appropriately supported by

sequence data of the ITS region. This work

represents the first molecular detection of P.

aleophilum by PCR assays in Iraq.


G

Haleem, Abdullah & Jubraeel: Morphological And Molecular Identification of Phaeoacremonium aleophilum…

A B

D

E

H

Fig. (1): Phaeoacremonium aleaophilum, A) Twenty- day old colony on MEA-left, and PDA-right B) Structures

on the surface of and in MEA. Adelophialide with conidia. C) Mycelium with Phialides. D) Conidiophores. E)

Type I phialide. F-G) Type II Phialides. H) Type III Phialides, I) Conidia. Scale bars: B,C, I=5µm; D – H =

10µm

6

I

F

C


Journal of Basrah Researches ((Sciences)) Volume 37. Number 4. E ((2011))

Fig. (2): Agarose gel electrophoresis 1% at 70 volt for 45 minutes. M represents unrestricted DNA as a

standard molecular weight marker. Lane1- 10 Whole Genomic DNA of P. aleophilum isolates isolated

from different locations of Duhok Governorate.

bp

1500

1000

900

800

700

600

500

400

300

M 1 2 3 4 5 6 7 8

Fig. (3). Agarose gel of the PCR products using primer pairs Pal1-Pal2. Lanes 1-3, P. aleophilum isolates

(DP1, DP2, and DP3). Lane 8, negative control of sterile distilled water; lane M, 1Kb Plus DNA Ladder.

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