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


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


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


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 


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


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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