Sugar beet is the most important sugar crop in Morocco. The main production areas of sugar beet are Doukkala, Tadla, Gharb, and Moulouya. Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV, genus Benyvirus), was first described in Italy and has spread in most sugar beet-growing areas of the world within a few decades. In the Tadla region, Rhizomania is one of the major phytosanitary problems for the sugar-beet industry. In 2010, five isolates from different locations in the Tadla region were collected for molecular characterization studies by sequencing the coat protein gene (CPg). The obtained results showed that all the sequenced isolates were in the B type of BNYVV and revealed the highest nucleotide similarity with the Belgian isolate B2 (AY696077), with a sequence homology of 100%. To the best of our knowledge, this is the first molecular characterization of BNYVV isolates in Morocco

Abdelmoughit AIT FRIHA

Adil ESSARIOUI

Ahmed AARABE

Bouchra CHEBLI

Imane BIBI

Mohamed AFECHTAL

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10 Aarabe et al.: Molecular characterization of Beet necrotic yellow vein virus infecting sugar beet
Molecular characterization of Beet necrotic yellow vein virus 
(BNYVV) infecting sugar beet in Tadla
A. AARABE1, I. BIBI2, B. CHEBLI1, A. ESSARIOUI3, A. AIT FRIHA4, M. AFECHTAL2 
(Reçu le 19/06/2017; Accepté le 26/01/2018)
Abstract
Sugar beet is the most important sugar crop in Morocco. The main production areas of sugar beet are Doukkala, Tadla, Gharb, 
and Moulouya. Rhizomania, caused by Beet necrotic yellow vein virus (BNYVV, genus Benyvirus), was first described in Italy 
and has spread in most sugar beet-growing areas of the world within a few decades. In the Tadla region, Rhizomania is one of the 
major phytosanitary problems for the sugar-beet industry. In 2010, five isolates from different locations in the Tadla region were 
collected for molecular characterization studies by sequencing the coat protein gene (CPg). The obtained results showed that all 
the sequenced isolates were in the B type of BNYVV and revealed the highest nucleotide similarity with the Belgian isolate B2 
(AY696077), with a sequence homology of 100%. To the best of our knowledge, this is the first molecular characterization of 
BNYVV isolates in Morocco.
Keywords: Rhizomania, sugar beet, molecular characterization, coat protein gene, Morocco.
1 School of Applied Sciences, B.P 1136, University Ibn Zohr, Agadir 80000, Morocco.
2 National Institute for Agricultural Research (INRA), Regional Center of Kénitra, Laboratory of Virology, Route de Sidi Yahya du Gharb, Km 9, B.P. 257, Kénitra.
3 National Institute for Agricultural Research (INRA), Regional Center of Errachidia.
4 National Office for Food Safety (ONSSA), Regional Direction of Casablanca-Settat, Plant Protection Service of El Jadida.
Corresponding author email: mohamedafechtal.inra@gmail.com
Résumé 
La betterave sucrière est la principale source de sucre au Maroc. Les principales zones de production de la betterave à sucre 
sont Doukkala, Tadla, Gharb, et Moulouya. La rhizomanie, causée par le virus des nervures jaunes nécrotiques de la betterave 
(BNYVV, genre Benyvirus), a été décrite pour la première fois en Italie et s’est propagée dans la plupart des régions productrices 
de la betterave sucrière. Dans la région de Tadla, la rhizomanie est l’un des principaux problèmes phytosanitaires majeurs pour 
la culture de la betterave sucrière. En 2010, cinq isolats de différentes localités de la région de Tadla ont été collectés pour des 
études de caractérisation moléculaire par le séquençage du gène de protéine de la capside (CP). Les résultats obtenus ont montré 
que les isolats marocains étaient dans le groupe B de BNYVV; ces isolats ont montré une grande similarité nucléotidique avec 
l’isolat Belge B2 (AY696077), avec une homologie de séquence de 100%. Au mieux de nos connaissances, il s’agit de la première 
caractérisation moléculaire des isolats de BNYVV au Maroc.
Mots-clés: Rhizomanie, betterave sucrière, caractérisation moléculaire, gène de la protéine de la capside, Maroc.
INTRODUCTION
Production of sugar beet is the most important sugar crop 
in Morocco. With an area of cultivation of 58.400 ha, it 
produces 3.6 million tons of roots (Anonymous, 1015). 
The main sugar beet production areas in Morocco are 
Doukkala, Tadla, Gharb, and Moulouya. Sugar beet pro-
duction is suffering from many problems, especially from 
diseases and pests. Very scarce information is available on 
viral diseases of sugar beet, especially on Beet necrotic 
yellow vein virus (BNYVV, genus Benyvirus), the causal 
agent of Rhizomania, which is one of the major phytos-
anitary problems for the sugar-beet industry worldwide. 
Heavy infection with rhizomania can cause a yield loss 
of up to 70% (Johansson, 1985) and a decrease in sugar 
content from 16-18% to less than 7% (Bongiovanni and 
Lanzoni, 1964).
BNYVV is transmitted by soil-borne plasmodiophorid 
(protozoan) vector, Polymyxa betae Keskin (Abe and 
Tamada, 1986; Tamada, 2002). The virus is cystosorus-
borne and survives many years in soils even without 
susceptible plants (Tamada, 2002). The disease appears as 
foci (patches) in low laying areas of sugar beet fields. The 
infected sugar beet plant shows mild to severe chlorosis 
particularly in vascular bundle, followed by venial necro-
sis (necrotic yellow vein). The virus also produces charac-
teristic symptoms on roots by exhibiting proliferation of 
lateral rootlets (rhizomania) and stunting (Tamada, 2002).
BNYVV has a multipartite single-stranded RNA genome 
with all natural isolates containing four RNA species, al-
though some isolates have an additional RNA-5 (Tamada 
et al., 1989). Three major serologically indistinguishable 
BNYVV groups named type A, B and P have been identi-
fied (Kruse et al., 1994; Koenig et al., 1995; Miyanishi et 
al., 1999; Koenig and Lennefors, 2000). BNYVV A type 
isolates are distributed worldwide, whereas the B type 
isolate is prevalent in limited areas of Europe, mainly in 
France and Germany, but occurs also in China and Japan 
(Miyanishi et al., 1999; Sohi and Maleki, 2004; Schirmer 
et al., 2005; Li et al., 2008). BNYVV A and B types typi-
cally contain only RNAs 1 to 4. BNYVV P type isolates 
that contain RNA-5 are detected only in France, Kazakh-
stan and in the UK (Koenig et al., 1997; Koenig and Len-
nefors, 2000; Harju et al., 2002; Ward et al., 2007). P type 
Caractérisation moléculaire du virus des nervures jaunes nécrotiques de la betterave (BNYVV) 
infectant la betterave à sucre au Tadla
11Rev. Mar. Sci. Agron. Vét. (2018) 6 (1): 10-13
isolates appear to be more virulent than both the A and B 
types (Heijbroek et al., 1999) and may evade activation 
of plant defense responses.
BNYVV was first reported from Italy (Canova, 1959) and 
Tamada and Baba (1973) identified the causal agent of this 
disease as a virus and later they called it BNYVV from the 
leaf symptoms it induces. It has since been reported from 
many sugar beet producing countries all over the world 
(Chiba et al., 2011). In Morocco, Rhizomania was first 
detected in 2004 in Tadla region, and then the presence of 
the virus was confirmed in Doukkala region (EPPO, 2005).
Although BNYVV has occurred in Morocco since 2004, 
no attempt has been carried out to characterize the local 
virus type. Therefore, the present study aims to character-
ize local BNYVV isolates.
MATERIAL AND METHODS
Five BNYVV sources were collected from different loca-
tions in the Tadla region (Afourer, Beni Amir, Dar Ouled 
Zidouh, Ouled M’Bark and Souk Sebt) and were used 
to perform RNA extractions using RNaesy Plant Mini 
Kit (Qiagen, Germany) according to the manufacturer’s 
instructions; then, tested by RT-PCR using specific prim-
ers to amplify a 567-bp fragment of the coat protein 
(CP) gene of BNYVV (Schirmer et al., 2005). The PCR 
program used was 94°C for 3 min, 35 cycles of 94°C for 
1 min, 61°C for 1 min and 74°C for 1 min, followed by 
final extension at 72°C for 10 min. PCR products were 
electrophoresed in 1.2% agarose gel in 1X TAE buffer and 
stained with ethidium bromide (25 ng/ml) and visualized 
on UV trans-illuminator. Fragment sizes were determined 
by comparison with a 1 Kb DNA standard marker (GeneR-
ulerTM DNA Ladder, 0.5 ug/ul).
PCR products corresponding to the CP (567 bp) gene, 
from five local BNYVV isolates form Tadla region (TDL1, 
TDL2, TDL4 and TDL5), were purified using a DNA 
extraction kit (Fermentas, Lithuania) and sequenced. 
Sequencing was carried out in two directions and each 
sample was sequenced twice. The obtained sequences 
were aligned using the BioEdit software version 7.0.9 
(Hall, 1999) and the MEGA software version 5.05 (Tamu-
ra et al., 2011). The CP gene sequence of one Moroccan 
isolate from Tadla region (RHZ-TDL-Mor) was deposited 
in the GenBank database under the accession number 
KY771166. RHZ-TDL-Mor was then confronted with 
other strain references available in the NCBI database. 
Calculation of pair wise nucleotide distances between 
sequences and clustering were performed using the phy-
logenetic and molecular evolutionary genetic analysis 
software MEGA version 5.05 for the estimation of the 
nucleotide homology (Tamura et al., 2011). 
Figure 1: Phylogenetic tree of Beet necrotic yellow vein virus encoded CP sequences using the neighbour-joining method. 
The names indicate the country of origin and name of the isolate and are followed by accession number (in parentheses). 
Scale bar indicates the phylogenetic similarity index (bar size refers to 0.005 amino acid changes per site). Numbers on 
branches represent the bootstrap values out of 1000 replicates. Only bootstrap values over 50 are shown.
12 Aarabe et al.: Molecular characterization of Beet necrotic yellow vein virus infecting sugar beet
RESULTS AND DISCUSSION
Multiple alignments and the sequence identity matrix of 
the CP gene showed a high sequence similarity among the 
five sequenced Moroccan BNYVV isolates in the Tadla 
region (100 % nucleotide identity). Thus, a low genetic 
diversity exists within the Moroccan BNYVV population; 
suggesting that the virus was not introduced into Morocco 
from multiple sources. The obtained results showed that 
the Moroccan isolate RHZ-TDL-Mor (KY771166) was in 
the B type of BNYVV and showed the highest nucleotide 
similarity with the Belgian isolate B2 isolate (AY696077), 
with a sequence homology of 100% (Figure 1). 
RHZ-TDL-Mor clustered with Belgian (AY696077) and 
Deutsch (AB56306) isolates indicating a possible com-
mon entrance or introduction from these regions. We 
also found that coat protein gene of local RHZ-TDL-Mor 
isolate was closely related to other sequences of different 
BNYVV isolates available in the database with a sequence 
homology ranging from 96.2% to 98.9%; supporting the 
idea that coat protein is a relatively stable sequence in 
BNYVV genome. 
Comparisons based on CP-encoding nucleotide sequences 
indicated that percentage identity is highly conserved for 
all the isolates reported worldwide, suggesting that either 
the virus has a very stable genome or this might be the 
incidence of a recent introduction of the virus in different 
sugar beet growing areas. The result is quite in agreement 
with the findings of Bouzoubaa et al., (1987) where high 
level of genome conservation was reported for BNYVV 
reported isolates. 
Rhizomania is one of the most economically important 
diseases affecting sugar beet, and is widely distributed in 
most sugar beet growing areas of the world. The studies on 
BNYVV worldwide revealed that there are three pathotypes; 
A-type, B-type, and P-type (Kruse et al., 1994; Koenig et 
al., 1995; Schirmer et al., 2005). The A-type is distributed 
throughout most sugar beet growing regions of the world and 
is, so far, the only form present in the Tadla region. 
Fields remain infested with BNYVV indefinitely by 
Polymyxa betae cystosori that remain dormant for up to 
25 years. It has been reported that the environmental con-
ditions and cultural practices do not alter the viability of 
the virus inside the resting spores (Tamada, 2002; Rush, 
2003). Therefore, rotation to non-host crops or lengthen-
ing rotations is ineffective at reducing disease incidence, 
and the only viable means of control has been natural host-
plant resistance. Indeed, the virus detection and timely 
diagnosis is of utmost importance.
CONCLUSION
BNYVV is one of the most economically important 
viruses infecting sugar beet in Morocco. Molecular char-
acterization studies showed low genetic diversity among 
BNYVV isolates in the Tadla region. Also, these local 
isolates were all of the BNYVV B type and showed the 
highest nucleotide similarity with the Belgian BE isolate. 
Results of the present study are important in terms of future 
efforts aiming at restraining the spread of BNYVV to new 
areas. As with other soil-borne viruses, efforts to eradicate 
BNYVV from newly contaminated fields could be very 
difficult if not impossible without abandoning cultivation 
of susceptible crops for years. In areas where BNYVV 
occurs, introduction of rhizomania-resistant sugar beet 
cultivars is the most important option whose sustainability 
depends on the genetic variability of the local BNYVV 
populations. This study provides information for planning 
future strategies to control rhizomania in Morocco.
Aspects which require further investigations include de-
termining the density of Polymyxa betae carry BNYVV, 
and the stimulation of cystosori in the soil to release 
zoospores in absence of the host or the needed trap plants 
to attract zoospores before planting sugar beet in infected 
soils. Additionally, more investigations are needed to de-
termine the predominant BNYVV type in the other beet 
sugar cultivation areas. Answers to these questions may 
help to develop control strategies against BNYVV and 
other viruses transmitted by plasmodiophorid vectors.
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Molecular characterization of Beet necrotic yellow vein virus (BNYVV) infecting sugar beet in Tadla

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Molecular characterization of Beet necrotic yellow vein virus (BNYVV) infecting sugar beet in Tadla

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