Chemical and Biological Control of Phomopsis amygdali the ... - Iresa

Chemical and Biological Control of Phomopsis amygdali the 

Causal Agent of Constriction Canker of Almond in Tunisia 

Ali Rhouma, Mohamed Ali Triki , Khaled Ouerteni, Institut de l’Olivier, Route de 

Soukra, Km 1,5, 3003 Sfax, Tunisia, and Meriem Mezghanni, Institut National 

Agronomique de Tunisie, Avenue Charles Nicolle, 1082 Cité Mahrajène, Tunis, 

Tunisia 

____________________________________________________________________________ 

ABSTRACT 

Rhouma, A., Triki, M. A., Ouerteni, K. and Mezghanni, M. 2008. Chemical and biological control 

of Phomopsis amygdali the causal agent of constriction canker of almond in Tunisia. Tunisian 

Journal of Plant Protection 3: 69-77. 

Almond trees (Prunus dulcis) growing in the Tunisian aeries were attacked by Phomopsis amygdali 

which causes constriction canker, a damaging and economically important fungal disease. The aim of 

this study, carried out in laboratory and in the field, was to test some fungicides and Trichoderma spp. 

antagonists. antagonists. Chemical experiments showed that benomyl, methyl-thiophanate and 

carbendazim were effective in completely inhibiting the mycelial growth and the germination of conidia. 

Field experiments revealed that except for procymidone, all tested fungicides significantly reduced 

disease compared to untreated control. Benomyl, methyl-thiophanate and carbendazim provided the best 

control and gave a more than 70% reduction in infected shoots. Biological experiments showed that the 

antagonists Trichoderma viride and Trichoderma harzianum reduced significantly the mycelial growth of 

P. amygdali. Field experiments revealed the occurrence of more than 50% reduction in infected buds as 

compared to the untreated control. 

Keywords: Almond, canker, fungicides, Phomopsis amygdali, Trichoderma 

____________________________________________________________________________ 

Constriction canker is a fungal disease 

that affects almond and peach trees 

growing in several aeries of the world and 

in the Mediterranean regions. The disease 

is caused by the fungus Phomopsis 

amygdali (17). The genre Fuscicocum has 

been replaced by Phomopsis according to 

the taxonomic studies carried out by Tuset 

and Portila (17). The pathogen was 

observed for the first time in California on 

Corresponding author : A. Rhouma 

roumaal@yahoo.fr 

Accepted for publication 30 May 2008 

almond (1), however it is found in 

Tunisian orchards on almond since 1968 

(16). 

The pathogen infects almond and 

peach twigs through fresh leaf scars in the 

fall and stipules, bud scale scars, blossoms, 

and fruit scars in the spring (2). However, 

direct infection through dormant buds has 

also been observed (18). Symptoms appear 

in early summer and become increasingly 

evident as more blighted shoots appear 

through late summer. Infected twigs and 

shoots wilt and die because of elongate, 

brown, sunken cankers, often with a zonate 

pattern, at their bases. Constrictions 

formed at the bases of infected shoots and 

Tunisian Journal of Plant Protection 69 

Vol. 3, No. 2, 2008

leaf symptoms produced well beyond the 

infection site result from a toxin named 

fusicoccin (3, 7). Gumming is commonly 

associated with cankers, but it is not a 

good diagnostic characteristic because 

other canker pathogens also cause 

gumming. Most fruit on infected shoots 

shrivel and drop off as the shoots 

desiccate. Shoots having more advanced 

cankers, usually as a result of fall 

infection, are often dead before or during 

bloom and fruit set. A quantitative survey 

in commercial New Jersey orchards 

conducted in 1997 and 1998 revealed yield 

losses of 28.5 and 21.0%, which translated 

into economic losses of $4,009 and $2,803 

per ha, respectively (8). 

A review of fungicides used against 

the fungi indicated that the mercurial and 

arsenic fungicides were effective in 

controlling the disease; however they were 

banned from use on crops as foliar sprays 

(3, 4). In order to substitute these products, 

dithiocarbamates and benzimidazoles were 

used but they were proved less efficient 

(4). Lalancette and Robison (9) 

demonstrated that chlorothalonil was the 

most efficient fungicide followed by 

captan, azoxystrobin and mycobutanil. As 

regards to the period spray, the lowest 

canker incidence and severity was obtained 

when fungicides were applied during both 

fall and spring. 

With the exception of these 

fungicides, few products are registered for 

use during the critical periods of host 

susceptibility (fall), and efficacy of several 

new active ingredients is currently 

unknown. Otherwise, in recent decades, 

elevated awareness of the impacts of 

pesticide use on the environment and 

human health has resulted in efforts to 

reduce reliance on chemical controls. 

Several countries have instituted more 

stringent regulation of pesticide 

manufacture, registration and use, thereby 

increasing the cost, and decreasing the 

availability of these tools. In many cases, 

the pests themselves have indicated the 

need for change, with pesticide resistance 

now a common reality in many weeds, 

insects and diseases. The need for 

alternatives to pesticides is imperative. 

Several reports indicated that biologically 

based technologies such as biological 

control could be more widely used to solve 

pressing needs in pest management. In the 

case of P. amygdali, little is known about 

the potential antagonistic effects of 

Trichoderma species. Since it is generally 

believed that a combination of biological 

and chemical control measures is 

necessary to minimize losses caused by 

plant pathogenic fungi, the major aim of 

the present study was to investigate the 

management strategy for constriction 

canker using biological and chemical 

treatments. Different actives ingredients 

(benomyl, carbendazim, methylthiophanate, 

procymidone, cuprocuivre) 

were applied during the fall and spring 

periods. As regards to the antagonists 

agents, three species were tested: 

Trichoderma viride, Trichoderma 

harzianum and a local species of 

Trichoderma. 

MATERIALS AND METHODS 

Fungal isolation and suspension 

preparation. P. amygdali was isolated 

from constriction canker of twigs sampled 

from the cultivar “Kossentiny”. It was 

grown from a single spore and maintained 

on Potato Dextrose Agar (PDA, 200 g of 

potato, 20 g of dextrose and 20 g of agar). 

Conidia were collected from 10 day-old 

cultures, suspended in 0,001% Tween 20 

(polyoxyethylene sorbitan monolaurate). 

They were then filtered through glass 

wool, washed three times with distilled 

water, and then re-suspended in water to 

give a final concentration of 5x10 4 

spores/ml,estimated by counting with a 

Malassez slide. 


Vol. 3, No. 2, 2008

Experimental design. A local almond 

cultivar (Kossentiny) located in a 

commercial orchard in the region of Bir 

Mellouli (Sfax, Tunisia) was chosen for 

study. Almond trees had histories of severe 

constriction canker. For each treatment, 

three homogeneous trees were selected 

with approximately 4 to 5 m in diameter 

and 3 to 4 m in high. For each almond tree, 

ten homogenous shoots were selected and 

five buds in each shoot were marked. The 

design employed was a randomized 

complete block with three replicates. 

Chemical treatments. Different 

fungicides were assayed for their activity 

against P. amygdali in vitro and in planta. 

Evaluated fungicides were: benomyl 

(Benlate 50), procymidone (Sumisclex, 

50WP), methyl-thiophanate (Pelt 44), 

carbendazim (Bavistin FL), metallic 

copper (Cuprocuivre 50WP). All 

fungicides were suspended in water and 

tested at 1/10, 1/5, 1, and 3/2 of the 

registration dose (RD). 

Laboratory studies. Germination essay 

was carried out in cavity slides. One 

hundred µl of the test suspension and 50 µl 

of the conidial suspension were pipetted 

onto cavity slides and then placed on 

Whatman N°1 filter paper in Petri dishes 

and held at 25°C, with a 12 h-photoperiod 

of approximately 145 µE/m 2 /sec. Counts 

of germinated conidia were taken 48 h 

post-incubation, and calculated means 

were based on 100 measurements. Four 

replications of each concentration, and two 

separate tests were performed. 

Fungicides were also tested for their 

activity against mycelial growth. 

Suspension of fungicides were prepared in 

sterile distilled water and added to PDA 

medium at approximately 50°C to yield to 

the appropriate concentration. After 

mixing, the amended PDA was dispensed 

into 9-cm-diameter Petri dishes and 

allowed to cool. Six-mm-diameter plugs of 

agar from actively growing medium of P. 

amygdali, grown on PDA, were placed 

with the surface mycelium face down on 

the medium. The plates were then 

incubated at 25°C, and mycelial growth 

was measured at 24 h intervals. Four 

replications of each concentration were 

used, and three separate tests were 

performed. 

Field studies. Fungicides were applied 

during both fall and spring periods. The 

disease assessment was carried out from 

mid-June trough the end of July by 

counting the number of constriction 

cankers and determining disease incidence 

and severity on the base of the percentage 

of infected shoots and the average number 

of cankers per shoot respectively. 

Biological Treatments. Biological 

control of Phomopsis canker was 

performed with the antagonists fungi 

Trichoderma viride, T. harzianum and 

Trichoderma sp. isolated in Tunisia. 

Laboratory experiments. In vitro tests 

were achieved by confrontation of isolates 

of Trichoderma and P. amygdali in Petri 

dishes. Six-mm-diameter plugs of agar 

from actively growing medium of P. 

amygdali and the antagonistic fungi were 

placed on PDA at a distance of 30 mm. 

Plugs were symmetrically of the dish 

center. The plates were then incubated at 

25°C, and mycelial growth was measured 

at 24 h intervals. Four replications of each 

concentration were used, and three 

separate tests were performed. The 

inhibition percentage of mycelium growth 

was determined according to the formula 

of Fokkema (5) and Sy (14): 

M% = 100 x (M 0 – M 1 )/M 0 , 

where M 0 was the mycelial growth of 

Phomopsis alone and M 1 the mycelial 

growth of P. amygdali in presence of the 

antagonist. 


Vol. 3, No. 2, 2008

Field experiments. Antagonists were 

applied during both fall and spring periods. 

Spore suspension of Trichoderma (10 6 

spores/ml) was deposed on the leaves 

scars. The disease assessment was carried 

out from mid-June trough the end of July 

by counting the number of constriction 

cankers and determining disease incidence 

and severity on the base of the percentage 

of infected shoots and the average number 

of cankers per shoot respectively. 

Statistical analysis. Analysis of 

variance (ANOVA) was performed using 

the general linear models (GLM) 

procedure of the SPSS software (version 

13) means were separated by Duncan test, 

and responses were judged significant at 

the 5% level (P = 0.05). 

RESULTS 

Favorable conditions for disease 

development occurred during the 

experiments essays. Fall and spring 

seasons had almost similar environment 

conditions except in spring 2002, when 

temperatures were warmer and rainfall was 

reduced as compared to 2003 and 2004. 

Chemical treatment. 

Effect of fungicides on germination. 

From Fig. 1 it can be seen that germination 

of conidia was completely inhibited by 

benomyl at its registration dose (RD) and 

even at 1/5 RD and 1/10 RD. This active 

ingredient was very active against 

germination. Copper showed the same 

activity against conidia germination, but its 

efficacy was observed only at higher 

doses. However, procymidone was unable 

to inhibit germination completely even at 

the highest dose. 

Effect of fungicides on mycelial 

growth of P. amygdale. In tests using 

fungicide-amended PDA, methylthiophanate, 

benomyl and carbendazim 

inhibited mycelia growth of P. amygdali at 

registered dose even at very low 

concentrations. However, complete 

inhibition did not occur even at the 

registration dose with copper and 

procymidone. Nevertheless, copper was 

more active than procymidone, at all tested 

doses (Fig. 2). 

70 

60 

Germination (%) 

50 

40 

30 

20 

10 

0 

Procy 

Be 

Fongicide 

Co 

1/10 

RD 

1/5 RD 

RD 

3/2 RD 

Fig. 1. Effect of fungicides on conidial germination of P. amygdali. Counts of germinated conidia were taken 48 h 

post-incubation. Each value is the mean based on 100 measurements. (RD: Registration Dose, Procy: Procymidone, 

Be: Benomyl, Co: Copper) 


Vol. 3, No. 2, 2008

7 

6 

mycelial growth (cm) 

5 

4 

3 

2 

1 

0 

Procy Co Carb Ben M-Thio 

RD 

3/2 RD 

1/10 RD 

1/5 RD 

Fongicide 

Fig. 2. Effect of fungicides on mycelial growth P. amygdali. Each value in the mean is based on 4 measurements in 

triplicate experiments. (RD: Registration Dose, Procy: Procymidone, Co: Copper, Carb: Carbendazim, M-Thio: 

Methyl-Thiophanate). 

Field studies. From the results 

illustrated in Fig. 3, related to field studies, 

it appears that except for procymidone, all 

the tested fungicides significantly reduced 

disease as compared with non treated 

control, but methyl-thiophanate and 

benomyl were equally effective in 

controlling Phomopsis canker and 

provided the best control. A 70% reduction 

in infected shoots was observed with 

theses fungicides. Besides, the number of 

constrictions cankers was significantly 

reduced. 

70 

a 

Biological treatment. 

Laboratory essays. The percentages of 

inhibition found with the different strains 

revealed that all tested species of 

Trichoderma reduced the mycelial growth 

of P. amygdali (Table 1). But, T. viride 

and T. harzianum appeared more efficient 

than the local species of Trichoderma. 

Field experiments. All tested species 

of Trichoderma reduced significantly the 

percentage of infected shoots as well as the 

number of constriction cankers (Fig. 4). 

1 to 2 cankers 3 to 4 cankers 


infected shoots (%) 

60 

50 

40 

30 

20 

10 

b 

b 

c 

c 

d 

0 

Control Ben M-Thio Carb Co Procy 

Fungicide 

Fig.3. Effect of fungicides on the disease incidence and severity of P. amygdali infection. (Ben: Benomyl, M-Thio: 

Methyl-Thiophanate, Carb: Carbendazim, Co: Copper, Procy: Procymidone). Bars with the same letter were not 

significantly different at the level 5%. 


Vol. 3, No. 2, 2008

Table 1. Inhibition of P. amygdali mycelial growth by different species of Trichoderma as 

compared with the non treated control 

Antagonists Inhibition of P. amygdali mycelial growth (%) 

T. viridae 25.00 b 

T. harzianum 23.57 b 

Local Trichoderma sp. 

18.14 a 

* Values followed by the same letter were not significantly different at the 5% level 

Infected shoots (%) 

70 

60 

50 

40 

30 

20 

10 

0 

a 



b b b 

Control T. viride T. harzianum T. sp 

Antagonist 

Fig. 4. Effect of antagonists on the disease incidence and severity of P. amygdali infection after treatments with 

different species of Trichoderma: T. harzianum, T. viride, and a local isolate of Trichoderma sp. Bars with the same 

letter were not significantly different at the level 5%. 

DISCUSSION 

P. amygdali has been observed for the 

first time in Tunisian orchards since 1968 

(16). Phomopsis canker causes severe 

attack particularly in wet autumn for some 

susceptible cultivars. Infections occur after 

leaves scars or bud break. In 2002, 2003 

and 2004, we have observed more serious 

damages in many orchards in the region of 

Sfax. Many fruit-growers have requested 

for help to control this fungal disease. 

Benomyl and methyl-thiophanate have 

been used with success in controlling 

many fungal diseases included Phomopsis 

canker. Our result showed that only 

benomyl, among the tested fungicides was 

effective in completely inhibiting conidia 

germination. Mycelial growth was 

inhibited by benomyl, carbendazim and 

methyl-thiophanate. Procymidone was 

inefficient in inhibiting conidia 

germination and mycelial growth, showing 

that this fungicide was apparently 

subjected to the problem of Phomopsis 

resistance. From these experiments, it also 

appeared that effectiveness of fungicides 

as inhibitors of conidial germination is not 

always indicative for their performance 

against mycelial growth. This situation 

was observed in the case of copper, which 

was unable to control mycelial growth as 

its performance in inhibiting conidial 

germination. 

Fields experiments carried out in an 

orchard naturally infected with Phomopsis 

canker revealed that benomyl together with 

methlyl-thiophanate and carbendazim 

significantly reduced the percentages of 

infected shoots and the number of 

constriction cankers as compared to the 

other fungicides. Although benomyl was 

proved to be efficient in reducing disease 

severity, it can not be recommended 

because it has been banned for use in some 

countries. These chemicals should be used 

as mixtures, intermittently, or alternately in 


Vol. 3, No. 2, 2008

order to ovoid the possible selection of 

resistant strains. According to Norman and 

Robison (12), the fall leaf abscission 

period is the most critical time for disease 

control. These authors have reported that 

chlorothalonil and captan provided the 

lowest canker incidence and severity, 

followed by azoxystrobin and 

myclobutanil. The efficacy of various 

fungicides was compared on detached 

peach shoots that were artificially 

inoculated with P. amygdali conidia and 

incubated in moist chambers for 21 days 

(10). The best control was achieved with 

the triazole bitertanol and result in 89% of 

healthy shoots. However, sulfur, lime 

sulfur, and iprodione failed to provide any 

control. Chlorothalonil treatment in that 

study provided good control, confirming 

the results of Norman et al. (12). 

The potential of biological control 

agents Trichoderma harzianum and T. 

viride to suppress plant disease has been 

evaluated on a number of crops including 

almond tree. Our results applied for 

Phomopsis canker on almond tree showed 

the effectiveness of theses antagonists in 

laboratory and in field experiments. 

Efficacy of these antagonists was a direct 

result of action of antagonists through one 

or more of the following mechanisms: 

antibiosis, competition or hyperparasitism 

(6, 15). Many substances could be secreted 

such us trichodermine and also volatiles 

substances (11). Mycoparasitism has been 

reported in species of Trichoderma (13). 

Combining different control methods 

was recognized for best control of 

Phomopsis canker. Removing twigs with 

dieback and cankers is of interest in case 

of heavy attack. Chemical and biological 

treatments should be applied in 

complementary with the cultural methods. 

AKNOWLEDGMENTS 

This work was supported by the budget of the 

Institut de l’Olivier in Tunisia.We Thank Mr. 

Makhlouf and Mr. M. Megdich for their technical 

assistance. 

____________________________________________________________________________ 

RESUME 

Rhouma A., Triki M. A., Ouerteni K., and Mezghanni M. 2008. Lutte Chimique et biologique 

contre Phomopsis amygdali agent causal de la maladie du chancre phomopsien de 

l’amandier en Tunisie. Tunisian Journal of Plant Protection 3: 69-77. 

L’amandier (Prunus dulcis) cultivé à grande échelle en Tunisie, est sensible à l’attaque du champignon 

Phomopsis amygdali, responsable d’un chancre qui provoque d’importants dégâts sur les plantes 

infectées. Cette étude, conduite au laboratoire et au champ, consiste à tester l’efficacité de quelques 

fongicides et espèces antagonistes du genre Trichoderma. Les essais chimiques ont montré une efficacité 

remarquable de bénomyl, méthyl-thiophanate et carbendazime en inhibant complète la croissance 

mycélienne et germination conidienne du champignon. Les tests conduits aux champs à base de 

fongicides ont révélé, à l’exception de procimidone, une réduction significative de la maladie en 

comparaison avec le témoin non traité. Bénomyl et carbendazime se sont montrés les plus efficaces avec 

un pourcentage de réduction des pousses infectées supérieur à 70%. Les essais de lutte biologique ont 

montré que les espèces antagonistes Trichoderma viride et Trichoderma harzianum ont réduit 

significativement la croissance mycélienne de P. amygdali. Les essais en plein champ ont révélé une nette 

réduction de plus de 50% des bourgeons infectés par rapport au témoin non traité. 

Mots clés : Amandier, chancre, fongicides, Phomopsis amygdali, Trichoderma 

____________________________________________________________________________ 


Vol. 3, No. 2, 2008

_____________________________________________________________________ 

ملخص 

رحومة،‏ علي ومحمد علي تريكي وخالد الورتاني ومريم مزغني.‏ 

المسبَب لمرض التق ‏َرح المخنصر 

المكافحة الكيميائيَة 

والبيولوجيَة 

للفطر 

.2008 

للوز في تونس.‏ 

Tunisian Journal of Plant Protection 3: 69-77. 

Phomopsis amygdali 

الكائن المسبب لمرض التَقرح 

قي تونس للإصابة بفطر 

تتعرَض أشجار اللوز 

المخنصر،‏ الذي يلحق خسائرا آبيرة في الأشجار المصابة.‏ وتهدف هذه الدراسة المخبريَة والحقليَة إلى تقييم مدى فاعليَة 

في مكافحة 

بعض المبيدات الفطريَة الكيميائية والمبيدات البيولوجيَة باستعمال أنواع مضادة تتبع الجنس 

المرض.‏ أظهرت التجارب الكيميائية أن آارربندازيم وثيوفانات الميثيل وبينوميل آانت ناجعة في تثبّيط النم ‏َو 

الغزلي/الميسيليومي وإنبات الأبواغ الكونيدية للفطر.‏ بيَنت التجارب الحقليَة أن آل المبيدات الكيميائية المذآورة،‏ باستثناء 

بروسيميدون،‏ خفضت نسبة المرض معنوياً‏ مقارنة بمعاملة الشاهد غير المعالج.‏ أدى استخدام آل من آاربندازيم وثيوفانات 

الميثيل وبينوميل إلى خفض درجة إصابة الأغصان بنسبة تفوق أظهرت التجارب البيولوجية باستعمال الكائنين 

أنهما خفضا معنويًا النمو الغزلي للفطر 

المضادَين 

آما بيَنت التجارب الحقليَة انخفاض عدد البراعم المتقرَحة وذلك بنسبة تفوق مقارنة بمعاملة الشاهد غير المعالج.‏ 

.P. amygdali 

Trichoderma 

Phomopsis amygdali 

%50 

.%70 

و Trichoderma harzianum 

(Prunus dulcis) 

Trichoderma viride 

آلمات مفتاحية:‏ تقرَح،‏ لوز،‏ مبيدات فطريَة،‏ 

Trichoderma ،Phomopsis amygdali 

____________________________________________________________________________ 

LITERATURE CITED 

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of fruit rot and associated branch dieback of 

almond in California caused by a Phomopsis 

species tentatively identified as P. amygdali. Plant 

Dis. 83 : 1073. 

2. Cohoon, D. F., and Daines, R. H. 1956. Peach 

canker (Fusicoccum amygdali) : times and sites of 

infections. Plant Dis. Rep. 40 : 304-308. 

3. Daines R.H. 1959. Advises Bordeaux-Arsenic 

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4. 

4. Daines, R.H. 1974. Peach canker (Fusicoccum 

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Hammonton area. Hortic. News New Jersey State 

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Vol. 3, No. 2, 2008

16. Trigui, A. 1968. Sur la présence en Tunisie de 

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status of Fusicoccum amygdali and Phomopsis 

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Schultheiss, R.A. 1997. Pathogenicity of a species 

of Phomopsis causing shoot blight on peach in 

Georgia and evaluation of possible infection 

courts. Plant Dis. 81: 983-989. 

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