Biological, Serological, and Molecular Characterization of ... - Iresa

Biological, Serological, and Molecular Characterization of 

Pepper mild mottle virus (PMMoV) in Tunisia 

Monia Mnari-Hattab and Kerim Ezzaier, Laboratoire de Protection des Végétaux, 

INRAT, Rue Hédi Karray, 2049 Ariana, Tunisia 

ABSTRACT 

Mnari-Hattab, M. and Ezzaier, K. 2006. Biological, serological, and molecular characterization 

of Pepper mild mottle virus (PMMoV) in Tunisia. Tunisian Journal Plant Protection 1: 1-12. 

In Tunisia, the main virus disease infecting protected pepper (Capsicum annuum L.) has been Tobacco 

mosaic virus (TMV) common strain. The F1 pepper hybrid J-27 carrying the L 1 resistance gene to 

TMV was obtained in a local breeding program and cultivated for several years. Severe necrosis on 

fruits and mottling on leaves have been observed. Two isolates called Sah.11-00 and Teb.24-00 were 

determined as Pepper mild mottle virus (PMMoV) on the basis of DAS-ELISA. Biological 

classification based on pathotype-genotype interaction demonstrated that local isolates, Sah.11-00 and 

Teb.24-00, reacted as PMMoV(1-2) specially by their ability to infect Capsicum species containing L 1 

and L 2 resistance genes and their inability to infect tomato species and Capsicum genotypes containing 

L 3 and L 4 genes. Antibodies produced against local isolate Sah.11-00 strongly reacted with its 

homologous antigens as well as with the isolate Teb.24-00 and reference strains of PMMoV. Crossreactions 

with TMV common strains were minimized after absorption with heterologous antigens. A 

modified procedure involving immunocapture (IC) followed by reverse transcription polymerase chain 

reaction (RT-PCR) in a single step reaction was developed for detection of PMMoV and differentiation 

of pathotypes using EcoR I restriction enzyme analysis (RFLP). This procedure was proven to be 

useful for diagnosis and will be helpful for epidemiological investigations. This new occurrence of 

such breaking-resistance isolates of PMMoV in Tunisia may create a new challenge for the national 

breeding program for multivirus resistance in pepper. 

Keywords: Pepper, PMMoV, Tobamoviruses, pathotype, resistance, antibodies, IC-RT-PCR, RFLP 

In Tunisia, pepper (Capsicum annuum 

L.) is grown in several areas starting from 

the North regions to the South in the 

oasis. The plains of the centre (Kairouan 

and Sidi Bouzid) and the east coastal area 

are the main production areas. Pepper is 

cultivated under cold plastic tunnels as an 

early crop from September to July, and as 

a full-season vegetable as an open field 

crop from March to November. Full 

season pepper crop is strongly affected by 

Potato virus Y and Cucumber mosaic 

virus (PVY and CMV) which are 

Corresponding author: M. Mnari-Hattab 

hattab.monia@iresa.agrinet.tn 

Accepted for publication 4 May 2006. 

estimated to cause up to 50%losses of 

potential production in pepper varieties 

cultivated in Tunisia (2, 14, 22). However 

these two viruses are less frequent under 

cold plastic tunnels (14). The main virus 

infecting protected culture is Tobacco 

mosaic virus (TMV) common strain (16). 

It affects strongly the yield and fruit 

quality of local cultivars Baker and 

Baklouti (14, 22). The F1 pepper hybrid 

(J 27) carrying the L1 resistance gene 

against the common strain of TMV and 

adapted to cold protected crop was 

obtained in a local breeding program (14) 

and cultivated for several years. 

Tobamoviruses were mentioned to cause 

significant economic losses on infected 

pepper in the world (1, 24) and especially 

Tunisian Journal of Plant Protection 1 

Vol. 1, No. 1, 2006

in Mediterranean area (12, 30). These 

Tobamoviruses causing Pepper mild 

mottle virus (PMMoV) disease have been 

ordered on the basis of their interaction 

with the L gene for resistance in pepper 

genotype. Based upon their ability to 

overcome the resistance conferred by 

allelic series of Capsicum spp. genes 

known as L 1 , L 2 and L 3 they have been 

designed as pathotypes P 1 , P 1,2 , 

respectively (11). In the Capsicum genus, 

four alleles of the Tobamovirus resistance 

gene L have been discovered. The allele 

L 4 confers resistance to the more 

aggressive pathotype PMMoV 1,2,3 which 

was originally found in some lines of 

Capsicum chacoense and was introduced 

in the Hungary pepper type “Himes F1” 

(24). 

In the last five years, severe necrosis 

on fruits and mottling on leaves similar to 

those caused by Tobamoviruses have 

been observed on the J27 F1 resistant 

hybrid cultivated in the central coastal 

area. Paying attention to these 

observations, we attempt in our study to 

identify and characterize this virus using 

biological, serological, and molecular 

tools. Host ranges, serological and 

molecular reactions of these isolates were 

compared to those of reference isolates. 

MATERIALS AND METHODS 

Virus isolates and strains. Two virus 

isolates Sah.11-00 and Teb.24-00 were 

taken in this study with reference isolates 

of four strains belonging to the 

Tobamovirus group, kindly supplied by 

Gebre-Selassie from INRA Avignon- 

France (1998). They are described in 

Table 1. The studied isolates were 

collected from J27 pepper hybrid plants 

grown under cold plastic tunnels during 

early season crop 2000/2001 in two 

regions Teboulba and Sahline situated in 

central coastal area of Tunisia. Severe 

Table 1. Strains and isolates used in this study 

Isolates Original host Country Authors 

ToMV: SM2 Tomato France Messiaen and Maison, 1964 (21) 

TMV: Vi 76 Pepper France Gebre-Selassié and collaborators (12) 

PMMoV (1-2): Adam Pepper France Gebre-Selassié and collaborators (12) 

PMMoV (1-2-3): Eve Pepper France Gebre-Selassié and collaborators (12) 

Sah.11-00 Pepper Tunisia This work 

Teb.24-00 Pepper Tunisia This work 

necrotic symptoms on fruits and mottling 

have been observed on leaves of J27 

infected plants. All reference and local 

isolates were long-term stored by 

desiccation of small amounts of leaf 

materials of pepper over calcium chloride 

(CaCl 2 ) at 4°C according to the method 

described by Bos (3). 

Biological tests. Fresh collected or 

calcium chloride conserved dried leaves 

were ground in 0.03 M phosphate buffer 

(pH 7.2) and containing 0.2% Sodium 

diethyldithiocarbamate (Na-DIECA) at 

the rate 1:4. Activated charcoal (75 

mg/ml buffer) and celite were added to 

the extract. Inoculation was made by 

rubbing the extract on 5-leaf stage plants 

for each isolate tested. The isolates were 

cloned after two successive passages on 

Nicotiana glutinosa. A single local lesion 

was used to propagate the virus in each 

time. Virus cultures were maintained in 


Vol. 1, No. 1, 2006

pepper J27 F1 hybrid and propagated on 

Nicotiana benthamiana. Inoculated 

pepper plants and other indicator plants 

were subsequently transferred into an 

insect proof glasshouse with temperature 

ranging from 25 to 28°C during day and 

from 12 to 15°C at night. Host range tests 

were performed with two to four plants 

per species (Table 3). Symptoms were 

scored four to five weeks after 

inoculation. Back inoculation to at least 

two plants of Datura stramonium or N. 

glutinosa from non-inoculated leaves was 

made. 

Serological tests. Serological assays 

were carried out using the standard 

sandwich DAS ELISA according to Clark 

and Adams (1977) on pepper samples 

showing virus like symptoms to check for 

common viruses TMV, PVY, CMV and 

for new virus infection with PMMoV. 

Indirect ELISA test was performed 

according to Lommel and collaborators 

(19) to determine the obtained antibody 

titer. The antibodies used to detect PVY 

were locally produced in the laboratory of 

plant protection at INRAT; those used to 

detect CMV were kindly supplied by Drs 

Marchoux and Gebre-Selassie from 

INRA Avignon. PMMoV sera were 

purchased from BIO-RAD Phytodiagnostics 

(France) and used according 

to the manufacturer’s conditions. 

Virus purification and antiserum 

production. The isolate Sah.11-00 was 

purified according to the method 

described by Gebre-Selassie and 

Marchoux (11). N. benthamiana was used 

for propagating the virus. Systemically 

infected leaves were homogenized in 0.5 

M phosphate buffer pH 7.2 (Na 2 HPO 4 - 

KH 2 PO 4 ) containing 1% (v/v) of 2- 

mercaptoethanol with the ratio of 100 g 

leaf material per 200 ml of buffer. The 

homogenate was clarified by adding 8% 

(v/v) n-butanol using centrifugation at a 

low speed centrifugation at 10,000 x g for 

30 min. PEG 6,000 and NaCl were added 

to the supernatant in order to give a final 

concentration of 3% and 1% (w/v), 

respectively. A second precipitation by 

PEG was performed with Triton X-100 

being added (5% v/v) to the supernatant. 

Virus particles were further purified by 

two cycles of differential centrifugation: 

90,000 x g for 90 min and 10,000 x g for 

15 min. The obtained pellet was 

resuspended in a 1 mM EDTA buffer, pH 

7.2. Purified virus was stored at –18°C 

until use. 

Virus concentration and purification 

yield were estimated using a specific 

extinction coefficient (A 0.1% 1 cm at 260 

ηm) = 3.18 (30). 

A rabbit was immunized by injecting 

0.5 mg of purified virus emulsified with 

an equal volume of Freund’s complete 

adjuvant. Four intramuscularly injections 

were performed at one-week interval and 

three more injections two week spaced. 

Bleedings started one month after the first 

immunization. 

Absorption of serum. IgGs of the 

obtained antiserum were purified by 

chromatography column sepharose CL- 

4B protein A (from Pharmacia) and 

absorbed according to Dijkstra and Jager 

(10) against virus-free plant material and 

SM2 TMV strain. Sap extracts from 

virus-free plants (N. benthamiana) and 

SM2 infected plants were squeezed 

through cheesecloth and diluted 20 times 

in PBS-T. Antibodies were diluted in the 

obtained plant sap. The mixture was 

incubated for one hour at 37°C and 

centrifuged at 8,000 x g for 20 min. The 

supernatant (absorbed antiserum) was 

used in indirect ELISA. 

Molecular tests. 

Oligonucleotide primers. In this 

investigation, the set of PMMoV specific 

primers P12/3 (5’-ACAgCgTTTggATCTTAgTAT-3’) 

and P12/3A (5’- 

gTgCggTCTTAATAACCTCA-3’) were 

used (28). 

IC-RT-PCR. Immunocapture (IC) was 

performed according to Wetzel and 

collaborators (31). Each PCR tube (0.2 ml 

Biozym) was incubated 3 hours at 37°C 

with 100 µl of PMMoV or TMV 

antiserum diluted in carbonate buffer pH 

9.6 as used in ELISA test, then washed 


Vol. 1, No. 1, 2006

three times with 150 µl of PBST at pH 

7.4 (137 mM NaCl, 1.5 mM KH 2 PO 4, 8 

mM Na 2 HPO 4, 2.7 mM KCl) containing 

0.05 % (v/v) Tween 20. The plant 

samples infected by PMMoV and TMV 

were ground in ELISA extraction buffer 

(PBST containing 2% (w/v) of 

Polyvinylpyrrolidon and 0.2 % (w/v) of 

bovine serum albumin). The homogenate 

was spun at 2,000 x g for 5 min at 4°C. 

Hundred microliters of the supernatant 

were added to coated tubes and submitted 

to an immunocapture phase for 2 hours at 

30°C. Then, they were washed 3 times 

with PBST buffer and one more with 

sterile bidistilled water. The detection 

method was based on one-tube RT-PCR 

assays using Titan kit from Roche 

diagnostics (Penzberg, Germany) 

according to manufacturer's conditions. A 

mix of 50 µl was prepared as following: 

0.2 mM of each dATP, dCTP, dGTP, 

dTTP. 0.4 µM of each primer; 5mM 

DTT; 10 µl of (5 x RT-PCR) buffer 

containing 1.5 mM MgCl 2 and 1 µl of 

AMV and Expand High fidelity PCR 

system. The volume of mix was adjusted 

with sterile bidistilled water and added to 

each coated tube. 

The amplification was carried out in a 

Biometra Uno II programmed for one 

cycle of 50°C for 30 min and a second 

cycle of 94°C for 2 min followed by 35 

cycles (denaturing at 94°C for 30 s, 

annealing at 50°C for 1 min and extension 

at 68°C for 1 min) and terminated with a 

final extension cycle at 68 °C for 10 min. 

IC-RT-PCR-RFLP. RFLP analysis 

was performed for Sah.11-00 and Teb.24- 

00 isolates and reference strains PMMoV 

(1-2) and PMMoV (1-2-3) by digesting 

the amplified product obtained from IC- 

RT-PCR with primers P12/3 and P12/3A 

with EcoR I enzyme according to 

manufacturers conditions. PCR products 

and digested fragments were run in a 1% 

(w/v) agarose gel contained ethidium 

bromide in TAE buffer (40 mM Trisacetate, 

1 mM EDTA) under a 100 mA 

and were visualized with a UV 

transilluminator at 312 nm. 

Absorbance at 405 nm 

RESULTS 

Purification and serology. Concentration 

of purified virus of isolate 

Sah.11-00 was estimated to 0.4 g/kg plant 

tissues. DAS-ELISA tests demonstrated 

that extracts from Sah.11-00 and Teb.24- 

00 infected plants strongly reacted with 

antiserum produced against PMMoV 

from Bio-Rad but neither against PVY, 

CMV nor TMV antiserum. The titre of 

our anti-PMMoV antiserum when tested 

by indirect ELISA was found to rise to 

10 –5 against homologous antigens and 

other PMMoV strains, 10 -4 against TMV 

strains Vi76 and SM2 whereas it was 

around 10 -3 against healthy plant sap (Fig. 

1). 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

10 -3 10 -4 10 -5 10 -6 10 -7 

Dilution of antibodies 

Sah.11-00 

PMMoV(1,2,3) 

PMMoV(1,2) 

SM2 

Vi76 

T(-):N. Benthamiana 

Fig. 1. Reactivities in indirect ELISA of purified 

antibodies at different concentrations with their 

homologous antigens (local isolate Sah.11-00), 

heterologous antigens (reference PMMoV strains 

and common strain of TMV) and with healthy plant. 

Absorbed antibodies with healthy 

plant still produce a good homologous 

reaction with Sah.11-00 and against 

PMMoV (1,2) but they still cross-reacted 

with the common strain of TMV (Vi 76 

and SM2) (Fig. 2). These absorbed IgGs 

were secondly absorbed with SM2. They 

still produce a good reaction with Sah11- 

00 but did not react positively with 

healthy plant or with SM2 infected plant 

(data not shown). 


Vol. 1, No. 1, 2006

A b s o r b a n c e a t 4 0 5 n m 

Reactions on various indicator 

plants. Sah.11-00 and Teb.24-00 isolates 

were inoculated as described previously 

on a range of indicator plants and at the 

same time to a pepper host range with 

known pathotype-genotype interaction of 

Tobamoviruses (Table 2). 

3.5 

3 

2.5 

2 

1.5 

1 

0.5 

0 

2x10 -3 10 -3 1/2x10 -3 1/4x10 -3 

Dilution of antibodies 

Sahline11-00 

T(-) 

Vi 76 

SM2 

PMMoV(1,2) 

Fig. 2. Reactivities in indirect ELISA of absorbed 

IgGs at different concentrations with their 

homologous antigen (Sah11-00), with strain 

PMMoV(1,2), with TMV common strain (Vi76 and 

SM2) and with healthy plant (Nicotiana 

benthamiana) 

Reactions of tested plants are 

summarized in Table 3. Three weeks after 

inoculation, our two isolates reacted as 

following: 

- D. stramonium, N. tabacum Xanthi–nc 

and N. glutinosa displayed small necrotic 

local lesions, 

- N. tabacum cv Samsun did not show 

neither local nor systemic symptoms and 

the virus could not be detected from 

newly developed leaves, 

- N. occidentalis displayed a systemic 

mosaic and leaf deformation, 

- N. benthamiana reacted with systemic 

chlorosis, leaf deformation and growth 

reduction, 

- Physalis floridana showed systemic 

mottling and some leaf deformation. 

Symptoms were more severe in the case 

of rise of temperature, 

- Lycopersicon esculentum F1 hybrid 

Razan was immune to the virus as proven 

by back-inoculation, 

- Solanum melongena on which only faint 

chlorotic local lesions appeared but the 

virus could not be detected neither from 

inoculated nor from non-inoculated 

leaves. 

Typing on various Capsicum 

genotypes. Reactions on Capsicum 

species were carefully observed in order 

to classify the isolates studied on the basis 

of virus-host genotype interactions. 

- On local pepper varieties Baker and 

Baklouti inoculated leaves were fallen 

down one week after inoculation, plants 

developed systemic chlorosis, mottling 

and leaf rugosity, some necrosis may 

appear on the stems. 

- Hybrid J27 showed no local symptoms, 

systemic infection started by vein clearing 

followed by systemic necrotic spots and 

mottling. 

- Hybrids Roda/Drago (L 1 ) and Greygo 

(L 2 ) displayed a systemic mottling two 

weeks after inoculation. 

- Hybrids Novi (L 3 ), Rapires (L 3 ), Cuzco 

(L 4 ), Himes (L 4 ) and C. chacoense 

PI260429 (L 4 ) displayed local necrotic 

lesions five days after inoculation which 

then started to enlarge and coalesce. The 

ultimate stage is the falling down of the 

inoculated leaves. No virus activity was 

detected on top leaves, i.e. no symptoms 

and no infection as verified by back 

inoculation. 

IC-RT-PCR for detection of 

PMMoV. IC-RT-PCR test was applied to 

common strain of TMV, to our isolates of 

PMMoV Sah.11-00 and Teb.24-00, to 

reference strains PMMoV (1-2), PMMoV 

(1-2-3), and to healthy sample. The size 

of the amplified product was as expected 

around 836 bp with local isolates and 

reference strains of PMMoV but neither 

with TMV (Vi 76) (lane 2) nor with 

healthy sample (lane 7) (Fig. 3). 

Differentiation of PMMoV strains 

with IC-RT-PCR-RFLP. RFLP analysis 

was performed to Sah.11-00, to Teb.24- 


Vol. 1, No. 1, 2006

00 isolates and PMMoV (1-2) and 

PMMoV (1-2-3) by digesting the 

amplified fragment obtained from IC-RT- 

PCR with primer pair P12/3 and P12/3A. 

Restriction digestion with EcoR I enzyme 

yielded one single fragment with 

PMMoV (1-2), Sah.11-00 and Teb.24-00, 

whereas it produces two fragments of 260 

bp and 570 bp with PMMoV (1-2-3) 

reference strain (Fig 4). 

Table 2. Pepper host range used in biological tests, genotypes and origins 

Host Genotype Origins 

C. annuum: cv Baker L 0 : Hamza, INRAT unpublished data 

C. annuum: cv Baklouti L 0 : Hamza, INRAT unpublished data 

Hamza, N., INRAT, Tunisia. 

C. annuum: J27 F1 Hyb L 1 : Hamza and collaborators (14) 

C. annuum: Greygo L 2 : Kalman and collaborators (17) 

C. annuum: Rapires F1 Hyb L 3 : Kalman and collaborators (17) 

Gáborjányi.,Vezprém university, 

Georgikon Faculty of 

Agricultural Science, Hungary. 

C. annuum: Himes F1 Hyb L 4 : Sagi and Salamon (24) 

C. annuum: Novi L 3 : Palloix, personal communication 

C. chacoense: PI 260429 L 4 : Boukema (4) and Gebre-Selassie and 

collaborators (12) 

Palloix,.A.,INRA-Montfavet. 

France. 

C. annuum: Roda/Drago 

(P824) F1 Hyb 

C. annuum: Cuzco (BK162) F1 

Hyb 

L 1 

L 4 

Syngenta 

1 2 3 4 5 6 7 8 

800 pb 

Fig. 3. One percent agarose gel 

electrophoresis analysis of IC-RT-PCR 

product obtained after amplification with 

primers pair P12/3 and P12/3A. IC-RT-PCR 

product from Vi76 (Lane 2); PMMoV (1-2) 

(Lane 3); PMMoV (1-2-3) (Lane 4); 

Sah.11-00 (Lane 5); Teb.24-00 (Lane 6); 

healthy sample (Lane 7). Molecular weight 

marker Gene Ruler 100 base pair ladder 

plus from Roche (Lanes 1 and 8). 


Vol. 1, No. 1, 2006

1 2 3 4 5 

800 pb 

570 bp 

260 bp 

Fig. 4. One percent agarose gel 

electrophoresis analysis of EcoR I 

restriction analysis of IC-RT-PCR 

product obtained after amplification 

with primers pair P12/3 and 

P12/3A. PMMoV(1-2) (Lane 2); 

Sah.11-00 (Lane 3);Teb.24-00 

(Lane 4) and PMMoV(1-2-3) (Lane 

5). Molecular weight marker Gene 

Ruler 100 base pair ladder plus 

from Roche (Lane 1). 

DISCUSSION 

DAS-ELISA tests showed that Sah.11- 

00 and Teb.24-00 isolates were 

determined as PMMoV. The antibodies 

produced against purified isolate Sah.11- 

00 were used in indirect ELISA tests. 

They reacted strongly with PMMoV 

strains, less with TMV, and only slightly 

with healthy plant. Our produced 

immunogen was probably not totally 

pure. Indeed, we did not perform any 

gradient step at the end of purification; 

this can explain the reaction against 

healthy plant sap. 

The obtained cross reaction against 

the common strain of TMV can be 

explained by the close serological 

properties of PMMoV to some viruses 

belonging to Tobamovirus group, mainly 

TMV and ToMV as reported by Wetter 

and Conti (29) and Gebre-Selassie and 

Marchoux (11). Serological identification 

of species, which are related, might 

lead to ambiguous results especially 

within the Tobamovirus genus. 

Differentiation problems resulted from 

heterologous reactions of antisera 

detecting conserved epitopes (18). Our 

results demonstrated that after 

absorption with virus-free plant, the 

obtained IgGs still produce a good 

reaction with their homologous antigens 

but reacted with TMV strains (Vi 76 and 

SM2) (Fig. 2). This absorption allowed 

to discard the antibodies produced 

against plant proteins. The double 

absorption with sap of healthy plant and 

with SM2 infected plant gave better 

reaction with Sah. 11-00 and minimize 

there activities with TMV common 

strains (data not shown). 

Subgrouping of PMMoV isolates is 

important for elucidation of PMMoV 

epidemiology and for practical detection 

of PMMoV pathotypes. Classification 

system based on virus-host genotype 

interactions in which pathogenicity or 

virulence of strain is expressed in Arabic 

numerals relating to L-genes resistance 

in Capsicum sp. was used (Table 2). 

According to the obtained results (Table 

3) and to the descriptions of Gebre- 

Selassie and collaborators (12) and 

Gebre-Selassie and Marchoux (11) 

Sah.11-00 and Teb.24-00 isolates 

behaved like the PMMoV pathotype 1-2, 

by their ability to infect Capsicum 

species J27 and Greygo hybrids F1 

carrying respectively L 1 and L 2 

resistance gene and their inability to 

infect L. esculentum genotype and 

especially by their capacity to induce 

hypersensitive reaction on Capsicum 

genotypes carrying L 4 genes (PI260429, 

Himes and Cuzco) and on Capsicum 

genotypes carrying L 3 genes (Rapires 

and Novi). 

RT-PCR methods have been 

reported for detecting plant viruses 

within many genera such as Potyvirus, 

Tobamovirus, Luteovirus and 

Cucumovirus (6, 7, 9, 13, 15, 18, 23, 26, 

28). RT-PCR-RFLP has been also used 

to differentiate pathotype of PMMoV 


Vol. 1, No. 1, 2006

Table 3. Comparative host range of local strains to reference strain 

Host range Genotype Sah.11-00 Teb.24-00 PMMoV (1-2): Adam 

Capsicum annuum cv Baker 

Capsicum annuum cv Baklouti 

Capsicum annuum cv J27 

Capsicum annuum Hyb F1 Drago/Roda 

Capsicum annuum Hyb F1 Greygo 

Capsicum annuum Hyb F1 Novi 

Capsicum annuum Hyb F1 Rapires 

Capsicum annuum Hyb F1 Himes 

Capsicum annuum Hyb F1 Cuzco 

Capsicum chacoense PI 260429 

Lycopersicon esculentum Hyb F1 Razan 

Nicotiana tabacum cv Xanthi- nc. 

Nicotiana tabacum cv Samsun 

Nicotiana benthamiana 

Nicotiana glutinosa 

Nicotiana occidentalis 

Physalis floridana 

Solanum melongena F1 cv Black enorma 

Datura stramonium 

L 0 

L 0 

L 1 

L 1 

L 2 

L 3 

L 3 

L 4 

L 4 

L 4 

- / S 

- / S 

- / S 

-/ S 

-/ S 

L / - 

L / - 

L / - 

L / - 

L / - 

-* / - 

L / - 

l /- 

-* / S 

L / - 

-* / S 

-* / S 

L / - 

L / - 

- / S 

- / S 

-/ S 

- / S 

- / S 

L /(-/s) 

L / - 

L / - 

L / - 

-* / - 

L / - 

l / (s/-) 

Explanation of symbols: local reaction /systemic reaction; L: necrotic local lesions; l: latent local infection; S: 

Systemic symptoms; s: latent systemic infection; -: no infection as verified by back inoculation. -*: infection not 

tested; ( ) symbols between brackets refer to inconsistent or erratic symptoms depending on growing conditions. 

-* / S 

L / -* 

-* / S 

-* / S 

L / - 

L / - 

(18, 26, 28). These methods based on 

RT-PCR encountered several problems 

in epidemiological research of the viral 

diseases. RNA extraction is troublesome 

and molecules are also readily degraded 

due to the ubiquitous presence of RNase. 

In contrast, the IC-RT-PCR method 

avoided the extraction of viral or plant 

total RNA, and was easily carried out in 

a single tube. Such procedures have been 

developed for detecting many plant 

viruses including Grapevine leafrollassociated 

virus 1, Pepino mosaic virus 

and Plum pox virus (5, 20, 25, 31). Here, 

we applied IC-RT-PCR and IC-RT-PCR- 

RFLP procedure respectively for 

detecting PMMoV and for pathotypes 

identification. In IC-RT-PCR, we found 

that a single strong band of about 830 

nucleotides could easily be amplified 

from plant saps containing PMMoV 

Tunisian isolates and reference isolates 

(Fig. 3). This result confirms those 

obtained by Velasco and collaborators 

(28) using total RNA in RT-PCR test. 

Results also showed that a single 

strong band of about 830 nucleotides was 

amplified from plant saps containing 

PMMoV(1-2) and local isolates, whereas 

two bands of about 570 and 260 bp where 

obtained from plant saps containing 

PMMoV(1-2-3) when digested with EcoR 

I (Fig. 4). From sequence alignment of 

fragments obtained using the primer pair 

(P12/3 and P12/3A); Velasco and 

collaborators (28) demonstrated that the 

pathotypes (1-2) and (1-2-3) are 

differentiated by the absence of EcoR I 

restriction site in the first one. Restriction 

patterns of EcoR I enzyme were then 

sufficient to differentiate pathotypes of 

tested species. Pattern shown in Fig. 4 fits 

well with the assignment of the local 

isolates and the reference strain 


Vol. 1, No. 1, 2006

PMMoV(1-2) into the pathotype (1-2). 

Whereas the pattern obtained with 

reference strain of PMMoV(1-2-3) 

demonstrated the presence of the EcoR I 

restriction site which confirms its 

assignment to pathotype (1-2-3). 

Moreover IC-RT-PCR-RFLP confirms 

that PMMoV reference isolates 

Adam and Eve belonged respectively to 

the pathotypes (1-2) and (1-2-3) as 

reported by Gebre-Selassie and Marchoux 

(11) using biological classification. 

Characterization of the two local isolates 

and the reference strains using both 

pathotyping and IC-RT-PCR-RFLP 

showed a good correlation between both 

methods. Both appeared to be reliable. 

However pathotyping was timeconsuming, 

whereas IC-RT-PCR-RFLP 

method was efficient, time saving and 

could be widely used for identification on 

a larger scale. 

Based upon biological, serological, 

and IC-RT-PCR-RFLP reactions, we can 

conclude that Sah.11-00 and Teb.24-00 

can be assigned as isolates belonging to 

the strain (1-2) of PMMoV. This finding 

demonstrates the occurrence of this 

breaking-resistance pathotype in pepper 

crops in Tunisia. To our knowledge this 

virus has not been described in Tunisia 

and our investigation reports for the first 

time the natural occurrence of PMMoV in 

this country. This new occurrence may 

create a new challenge for the Tunisian 

breeding program for multivirus 

resistance in pepper (14). 

To get more information concerning 

the prevalence of PMMoV pathotypes, 

continuous surveys from year to year in 

different crops and locations will be 

required. The possible presence of 

pathotype (1-2-3) must be checked on a 

larger scale in Tunisian farms in order to 

pertinently introduce efficient resistant L 3 

gene against PMMoV (1-2). The 

introduction of suitable resistance gene in 

plants will offer a powerful and accurate 

tool to avoid PMMoV (1-2) infection 

especially in early-protected pepper crops 

of central coastal regions and possibly in 

regions where this virus could be 

identified. 

ACKNOWLEDGEMENTS 

Authors gratefully acknowledge Drs. Gebre- 

Selassie and Georges Marchoux (INRA Montfavet) 

for kindly providing reference strains and CMV 

antibodies, Drs. Alain Palloix (INRA Montfavet), 

R. Gaborjanyi, and G. Kazinczi (University of 

Veszpraém, Hungary) for kindly providing 

Capsicum species carrying resistance gene and Dr. 

Naceur Hamza (INRA Tunisia) for kindly 

providing local pepper varieties and hybrids. 

RESUME 

Mnari-Hattab M. et Ezzaier K. 2006. Caractérisation biologique, sérologique et moléculaire du 

virus de la marbrure légère du piment (PMMoV) en Tunisie. Tunisian Journal Plant Protection 

1: 1-12. 

En Tunisie, la souche commune du virus de la mosaïque du tabac (Tobacco mosaic virus TMV) sur 

piment (Capsicum annuum L.) est la cause de la maladie virale la plus répandue en culture protégée. 

L’hybride F1 J-27 porteur du gène de résistance L 1 vis-à-vis des souches communes de TMV a été 

obtenu dans le cadre d'un programme d’amélioration du piment en Tunisie et a été cultivé pendant 

plusieurs années. Des symptômes de nécroses sévères sur les fruits et des mosaïques sur les feuilles ont 

été observés. Deux isolats appelés Sah.11-00 et Teb.24-00 ont été identifiés en ELISA en utilisant un 

kit d’anticorps commercial spécifique vis-à-vis du virus de la marbrure légère du piment (Pepper mild 

mottle virus PMMoV). La caractérisation biologique basée sur l’interaction pathotype-génotype 

démontre l’appartenance des isolats tunisiens au PMMoV pathotype (1,2). En effet, les isolats Sah.11- 

00 et Teb. 24-00 infectent les génotypes du piment porteurs des gènes de résistance L 1 et L 2 et n’infecte 

pas ceux porteurs des gènes L 3 et L 4 ainsi que l'espèce de tomate. Un antisérum a été préparé à partir de 

l’isolat Sah.11-00. En ELISA, les anticorps obtenus détectent l’antigène homologue, l’isolat Teb.24-00 

et les antigènes des souches de référence du PMMoV. L’élimination par absorption des anticorps 


Vol. 1, No. 1, 2006

éagissant avec les extraits de plantes saines a permis d’obtenir un réactif très fiable de détection du 

PMMoV. Une procédure modifiée basée sur l’immunocapture (IC) suivie d’une RT-PCR en une seule 

étape a été développée pour l’identification du PMMoV et la différentiation des pathotypes en 

analysant les fragments obtenus après RFLP. Cette procédure, fiable, rapide et facile à mettre en 

pratique pour le diagnostic, pourrait être d’une grande utilité pour les études épidémiologiques futures. 

La nouvelle présence en Tunisie du pathotype 1-2 du PMMoV devra obligatoirement être prise en 

compte par les sélectionneurs et se traduire par une réorientation de leur programme d’amélioration du 

piment pour la résistance aux maladies virales. 

Mots clefs: Piment, PMMoV, Tobamovirus, pathotype, résistance, anticorps, IC-RT-PCR, RFLP 

ملخص 

مناري-حطاب،‏ منية وآريم الزائر.‏ التشخيص البيولوجي والسر ولوجي وعبر البيولوجيا الجزئية 

التبقع المرمري الخفيف للفلفل (PMMoV) في تونس.‏ 

لفيروس 

.Tunisian Journal of Plant Protection 1: 1-12 

.2006 

تعتبر السلالات العادية لفيروس تبرقش الدخان (TMV) السبب الرئيسي للأمراض الفيروسية للفلفل 

(.L annuum الأآثر تواجدا في البيوت المحمية في تونس.‏ أنتج الهجين J27 الحامل لجين المقاومة الفيروسية 

للسلالات العادية لفيروس TMV خلال برنامج وطني لتحسين الفلفل بتونس ووقع إآثاره طيلة سنوات عديدة.‏ آما 

لوحظت أعراض موت موضعي حاد على ثمار الفلفل و تبرقش على الأوراق.‏ فوقع التوصل إلى عزل وتشخيص سلالتين 

من فيروس التبقع المرمري الخفيف للفلفل (PMMoV) وذلك بطريقة تفاعل الأمصال 

سميتا 

ELISA باستعمال مجموعة تجارية للأجسام المضادة خاصة بتشخيص هذا الفيروس.‏ اظهر التشخيص الببيولوجي 

المعتمد على تفاعل النمط المرضي مع النمط الوراثي،‏ أن السلالات التونسية تنتمي إلى صنف 

،PMMoV حيث تصيب الفلفل الحامل لجينات مقاومة ولا تصيب الفلفل الحامل لجينات آما أنها لا 

تصيب نوع الطماطم.‏ وقع تحضير مصلا مضادا باستعمال سلالة وباعتماد طريقة ،ELISA تبين أن 

الأجسام المضادة لهذه السلالة تكشف على المادة الوراثية المماثلة على سلالة Teb24-00 وعلى المادات الوراثية 

للسلالات المرجعية لفيروس .PMMoV ومكًّن استبعاد الأجسام المضادة المتفاعلة مع مستخرجات النباتات السليمة من 

الحصول على مستحضر صالح لتشخيص فيروس .PMMoV ووقع استعمال طريقة بيولوجيا جزيئية محورة تعتمد على 

طريقة الحبس المناعي (IC) تليها طريقة RT-PCR في مرحلة واحدة لتشخيص فيروس PMMoV والتفريق بين 

الأنماط المرضية بتحليل الشظايا المتحصل عليها بعد استعمال طريقة .RFLP ويمكن أن تكون هذه الطريقة الدقيقة 

والسريعة والسهلة الاستعمال في التشخيص مفيدة جدا في الدراسة الوبائية لهذا الفيروس مستقبلا.‏ وتجدر الإشارة إلى 

وجوب الأخذ بعين الاعتبار لدى محسّني النبات بتونس،‏ تواجد هذا النمط المرضي لفيروس PMMoV(1,2) حتى يقع 

إعادة توجيه برامجهم البحثية في مجال التحسين الوراثي للفلفل في مقاومة الأمراض الفيروسية.‏ 

(Capsicum 

L 1 

(1,2) لفيروس 

و L 4 L 3 

.Sah11-00 

و L 2 L 1 

و Teb24-00 Sah11-00 

آلمات مفاتيح:‏ فلفل،‏ ،PMMoV توباموفيروس،‏ نمط ممرض،‏ مقاومة،‏ أجسام مضادة،‏ RFLP ،IC-RT-PCR 

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