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African Journal of Agricultural Marketing ISSN 2375-1061 Vol. 8 (10), pp. 001-009, October, 2020. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 
 

 

Full Length Research Paper 

 

Evaluation of cassava varieties for cassava mosaic 

disease resistance jointly by agro-inoculation 

screening and molecular markers 
 

Huiping Bi1, Mahender Aileni1,2 and Peng Zhang1* 
 

1
National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai 

Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China. 
2
Plant Biotechnology Research Unit, Department of Biotechnology, Kakatiya University, Warangal–506 009, India. 

 
Accepted 25 May, 2020 

 
The whitefly-transmitted cassava mosaic disease (CMD) has become a potential threat to cassava 
(Manihot esculenta Crantz) cultivation in ASEAN countries because of its devastating impact on 
cassava and overgrowth of whitefly vector regionally. To reduce the risk caused by the disease, it is 
necessary to evaluate the capacity of major cassava germplasms for CMD resistance to guide local 
farmers in adopting CMD-resistant cultivars once CMD epidemics occur. After agro-inoculation 
mediated infection of plantlets of collected cassava cultivars from China, Thailand and other ASEAN 
countries, the 18 cultivars tested developed various levels of CMD symptoms, indicating a lack of 
resistance to CMD. There was a positive association between symptom severity scores and 
accumulation levels of viral DNA in the different cultivars tested. The molecular markers RME1, SSRY28 
and/or NS158, which link with the CMD resistance loci CMD2 in cassava, were found in only three 
cultivars (11Q, T7 and N13) with moderate resistance to CMD. Our study suggests that CMD-resistance 
germplasms should be introduced from Africa. 

 
Key words: Manihot esculenta Crantz, cassava mosaic disease, agro-inoculation, molecular marker, screening. 

 
INTRODUCTION 

 
Cassava mosaic disease (CMD) is one of the major 
constraints in cultivation of cassava (Manihot esculenta 
Crantz), an important tropical and subtropical root crop 
(Fauquet and Fargette, 1990) . It is caused by several 
cassava mosaic geminiviruses and is the most important 
disease of cassava in Africa and the Indian subcontinent 
(Legg and Fauquet, 2004). Although there are no reports 
of cassava mosaic virus in ASEAN countries yet, there is 
a potential and serious threat on cassava production due 
to epidemics of the whitefly (Bemisia tabaci) vector in the 
cassava growing regions, which makes the producers 
and breeders to be at alert. 

Cassava, native to South America, was introduced to 

South China from surrounding South-East Asian countries 

countries 200 years ago (Zhang et al., 1998). In the last  
 
 
 
*Corresponding  author.  E-mail:  zhangpeng@sibs.ac.cn.  Tel: 
+86-21-54924096. Fax: +86-21-54924318. 

 
 
 
 
two decades, millions of cassava hybrid seeds provided 
by collaborators from the Centro Internacional de 
Agricultura Tropical (CIAT) with the support of Nippon 
Foundation were used for breeding new cultivars suitable 
for Chinese ecological environments. For example, 
cultivars SC5, SC8, GR891 and GR911 were bred in the 
farmer participatory crop improvement program (Howeler, 
2001). Recently, importation of core cassava germplasms 
from CIAT diversified the genetic background of cassava 
in China, allowing cassava breeders to produce new 
varieties with more desirable traits. Nevertheless, most of 
these germplasms are expected to be susceptible to 
CMD due to their South American origin. High CMD 
resistant cassava had been developed through 
integration of resistance trait from Manihot glaziovii by 
interspecific hybridization, which becomes the major 
resistance source dominating CMD resistance in pan-
Africa (Fargette et al., 1996). Classical genetic analysis 
and genetic mapping of some resistant landraces showed 
that a major dominant gene, CMD2, confers resistance to 

file:///C:\Users\user\Documents\REPUBLICATION\AGRICULTURAL%20SCIENCES\AppData\Local\Temp\www.internationalscholarsjournals.org


 
 
 

 

CMD and important molecular markers associated with 
CMD2 have been identified (Fregene et al., 1997; Akano 
et al., 2002). Another CMD resistance gene CMD1 was 
also identified and mapped on the cassava genetic 
linkage map by CIAT (Akano et al., 2002). CMD2 is 
located on linkage group R, whereas CMD1 is on linkage 
group D of the cassava molecular map (Fregene et al.,  
1997). The action of the two genes is also different: 
CMD2 is dominant, whereas CMD1 appears recessive in 
that its effect is detected only in backcross progeny and 
not in the F1 (Akano et al., 2002). CMD2 is on linkage 
group R of the male cassava framework map 8 cM from 
the marker SSRY28. Later, the group at CIAT identified 
NS158 to be closely linked to CMD resistance. The 
dominant CMD2 gene for CMD resistance was 
introgressed into Latin American germ- plasms through 
maker-assisted selection (MAS). After two years of 
evaluation, 14 genotypes with high CMD resistance and 
good rooting yield potential were selected (Okogbenin et 
al., 2007).  

In parallel, transgenic approaches of engineering CMD 
resistance in cassava have been reported (reviewed by 
Vanderschuren et al., 2007a). For example, increased 
ACMV resistance in cassava have been developed in tra-
nsgenic cassava plants expressing antisense RNA or 
dsRNA targeting the viral mRNAs of Rep (AC1), TrAP 
(AC2) and REn , or the viral untranslational common 
region (Zhang et al., 2005; Vanderschuren et al., 2007b, 
2009).  

Currently, a dozen of elite cassava varieties, for 
example KU50 and NZ199, are grown by local farmers in 
ASEAN countries. Evaluation of their capacity for CMD 
resistance is of importance due to devastating impact of 
CMD on cassava production. In order to screen cassava 
genotypes for CMD resistance efficiently, a platform that 
could integrate virus infection tests and marker-assisted 
selection would be useful. Most of ACMV inoculations 
were conducted by biolistic viral DNA delivery (Briddon et 
al., 1998; Zhang et al., 2005) and only recently, a very 
efficient agro-inoculation protocol with ACMV-NOg infe-
ctious clones in the laboratory was developed 
(Vanderschuren et al., 2009). In this paper, we developed 
a screening system, which combines the infection assay 
and CMD resistance-associated molecular markers to 
identify CMD resistant or susceptible cassava varieties. 
To our knowledge, this is the first report of screening 
CMD resistance using cassava varieties from ASEAN 
region. 
 

 
MATERIALS AND METHODS 
 
Plant material 
 
Cassava (M. esculenta Crantz) varieties used in the present study 
were grown in a greenhouse condition under 25 ± 2°C and 16 h/8 h 
photoperiod. Cassava varieties SC5, SC8, SC124, SC205, NZ199 
and ZM9781 were provided by Chinese Academy of Tropical 
Agricultural Science. Cultivar KU50 (Thailand) and GR911 were 

 
 

 
 

 
provided by Guangxi Institute of Subtropical Crops. Other cultivars 
including N13, Q12, R9, T7, V5, 3C, 5E, 11Q, 16P, 18R and 
TMS60444 were from the in vitro germplasm bank established in 
Shanghai Center for Cassava Biotechnology, Chinese Academy of 
Sciences. Besides the varieties mentioned above, two other 
varieties were also used in molecular marker analysis: TME3 (a 
Nigerian landrace displaying strong CMD resistance, as the positive 
control) and TMS30555 (a CMD-susceptible line, as the negative 
control) (Akano et al., 2002), kindly provided by International 
Institute of Tropical Agriculture (IITA). 

 

Virus isolate and bacterial strain 
 
The infectious clones of ACMV-NOg DNA-A and DNA-B (Liu et al., 
1997) harboring in the Agrobacterium tumefaciens strain LBA4404 
were used for agro-inoculation of all cassava varieties. The 
Genbank accession numbers of the published ACMV-NOg DNA-A 
and DNA- B sequences used in this study are AJ427910 and 
AJ427911, respectively. The construction of infectious clones of 
ACMV-NOg had been reported by Vanderschuren et al. (2009). 

 

Agro-inoculation 
 
Clones of A. tumefaciens strain LBA4404 containing the infectious 
clones of ACMV-NOg DNA-A and DNA-B (Vanderschuren et al., 
2009) were separately cultured on YEB plates supplemented with 
streptomycin (100 mg/l), rifampicin (25 mg/l) and kanamycin (50 
mg/l) at 28°C for 72 h. Then the bacteria of DNA-A and DNA-B 
were mixed together in equal proportion. One-month-old cassava 
plantlets with 5 - 6 leaves were needle-punctured 6 times with the 
mixed bacteria suspension at the tip of the shoot axis using a fine 
syringe needle (Ø 0.5 mm). A minimum of three plants per variety 
were inoculated. 

 

Molecular marker analysis 
 
Genomic DNA was isolated from fresh and young leaves of all 
uninfected cassava varieties grown in the greenhouse according to 
Dellaporta et al. (1983) . SSR markers SSRY28 and NS158, and a 
SCAR marker RME1 associated with CMD resistance were 
provided by Dr. Martin Fregene, CIAT, and were used in genotyping 
the DNA samples (Akano et al., 2002). Each 50 l PCR reaction 
contained 50 ng genomic DNA, 0.4 M of each forward and reverse 
primers, 10 mM Tris–HCl (pH 8.5), 50 mM KCl, 1.5 mM MgCl2, 0.2 
mM of each dNTP and 1 U Taq DNA polymerase. The PCR profile 
involved an initial denaturation for 5 min at 95°C; then 30 cycles at 
95°C for 40 s, 56°C for 1 min, 72°C for 30 s; and a final extension at 
72°C for 10 min. For SSR marker, 2.0 l of the PCR product was 
electrophoresed on 8% polyacrylamide gels for 5 - 6 h at 100 V, 
and DNA was visualized by standard silver staining using the 
Promega's silver staining kit (Promega, Shanghai, China). The 
SCAR marker was scored on a 1.5% agarose gel and visualized by 
ethidium bromide. 
 
 
Virus detection in infected plants using Southern blot analysis 
 
Total DNA was extracted from the top leaves of three infected 
plants per variety at 60 days post infection (dpi) according to Soni 
and Murray (1994). Aliquots of 5 ug of total DNA from each sample 
without digestion were analyzed using standard protocol for 
Southern blot (Sambrook et al., 1989). Accumulated viral DNA was 
hybridized with a DIG-labeled probe specific to the ACMV-NOg AC1 
gene. Labeling, hybridization and chemiluminescent detection were 
performed according to the instruction of the manufacturer (Roche 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 1. Cassava mosaic disease symptoms on plant and leaves of different cassava varieties after agro-

inoculation with African cassava mosaic virus infectious clone ACMV-NOg. A, B: TMS60444; C, D: KU50; 

E, F: Q12; G, H: SC8. 
 

 
Applied Science). The intensity of bands was quantified by using 

Eagle Eye® II still video system (Stratagene). 

 

CMD symptom assessment 
 
After agro-inoculation, CMD symptom development on new 
emerging leaves was assessed weekly. The symptoms were 
recorded from 10 dpi to 60 dpi till each plant has developed more 
than 20 new leaves. Disease symptom severity on fully expanded 
leaves was recorded on a scale of 0 - 4 (0, no symptom; 1, faint 
mosaic; 2, yellow mosaic, malformation, 5 - 10% size reduction; 3, 
severe mosaic, distortion, up to 50% size reduction; 4, severe 
mosaic, severe distortion, leaf reduced to veins with 50 - 80% size 
reduction), as described by Zhang et al. (2005). 
 

 

RESULTS 
 
Screening of cassava varieties for CMD resistance by 

agro-inoculation 
 
After agro-inoculation of 18 cassava varieties with ACMV-
NOg in a greenhouse, symptom development was monit-
ored regularly. Various foliar mosaic symptoms were dis-
played by all plants tested of TMS60444, KU50, Q12 and 
SC8 at 60dpi (Figure 1). Among these varieties, Q12 and 
SC8 showed more severe symptoms than TMS60444 
and KU50 at the whole plant level (Figure 1 A, C, E, G). 
Also, leaves of TMS60444 showed downward curling in 
most cases (Figure 1B) and KU50 displayed certain 
upward curling (Figure 1D). Yellowing, mosaic symptoms 
and reduced size were observed on the leaves of Q12 
(Figure 1E, F) whereas SC8 displayed foliar mosaic 
symptoms with different degrees of malformation vary 
from faint to severe (Figure 1G, H). These four varieties 
showed varied degrees of foliar symptom severity based 
on leaf position (Figure 2). Symptoms were most 

 
 

 

severe on leaves in positions of 7 - 9 for TMS60444, 
KU50, Q12 and SC8. For TMS60444, the average level 
of severity score had never reached 2.0 on newly 
developed leaves while the average level remained high 
for new leaves of Q12 and SC8. Symptoms observed on 
SC8 and Q12 are more severe than those of TMS60444 
and KU50. The symptom trend lines of other varieties are 
provided in the supplementary data (Suppl. Table 1).  

Total symptom severity scores of the 18 varieties are 
presented in Figure 3 in the order from lowest to highest 
(Figure 3, upper panel). Cultivar TMS60444, the model 
cultivar for genetic transformation frequently used in virus 
resistance study (Zhang et al., 2005; Vanderschuren et 
al., 2007b, 2009), showed a moderate resistance to 
ACMV- NOg infection. This is in consistent with results 
from a previous study on AFLP analysis of CMD resis-
tance (Fregene et al., 2000). In this study, all varieties 
were categorized into three groups: moderately resistant 
(MR) varieties R9, V5, 16P, TMS60444, N13, T7 and 11Q 
with total severity scores less than 40; moderately susce-
ptible (MS) varieties 3C, SC205, KU50, ZM9781, SC5, 
18R and GR911 with total severity scores from 40 - 50; 
and highly susceptible (HS) varieties Q12, SC8, SC124 
and NZ199 with severity scores more than 50. 
 

 

Viral DNA accumulation in infected plants 

 

The accumulation of ACMV DNA was detected by 
Southern blot analysis in apical leaves of inoculated 
plants at 60 dpi (Figure 4). In the six varieties analyzed, 
the accumulation of ACMV DNAs, including double-
stranded (ds), single-stranded (ss) DNA and open circular 
(oc) forms, was detected. The level of viral DNA 
accumulation was lower in N13, T7 and TMS60444 than 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 2. Cassava mosaic disease symptom severity scores on new emerging leaves 1 to 20 of cassava varieties 
KU50, SC8, Q12 and TMS60444 after ACMV-NOg agro-inoculation. The bottom panel illustrates representative 
leaves showing different degrees of symptoms for the evaluation of symptom severity scores (0 = no symptoms, 4 = 
severe symptoms). 

 

 

in KU50, SC8 and Q12. This result reflected a correlation 

between symptom severity and level of viral DNA 

accumulation. 
 

 

Molecular marker analysis 

 

The 18 cassava varieties, together with the CMD 
resistant line TME3 and the susceptible line TMS30555, 
were evaluated by three markers associated with the 
CMD resistance gene CMD2, including sequence-
characterized amplified regions (SCAR) RME1 and 
simple sequence repeats (SSR) SSRY28 and NS158 
(Lokko et al., 2007). For the SCAR marker RME1, only 
varieties 11Q (Suppl. Figure 1A, lane 11) and T7 (Suppl. 
Figure 1A, lane 18) showed the expected bands, similar 
to the CMD resistant line TME3 (Suppl. Figure 1A, lane 
21). Resistance-associated bands were obtained using 
primers of SSR marker SSRY28 in varieties N13, 11Q 
and T7 (Suppl. Figure 1B; lanes 9, 11, 18, respectively); 

 
 

 

whereas varieties 11Q (Suppl. Figure 1C, lane 11) and 
T7 (Suppl. Figure 1C, lane 18) showed the same pattern 
as the resistant line TME3 by using SSR marker NS158. 
Using SCAR-RME1 and SSR-NS158, variety 18R (Suppl. 
Figure 1A and C, lane 7) showed a similar pattern as the 
susceptible line TMS30555 (Suppl. Figure 1A and C, lane 
22).  

From the molecular marker analyses and agro-
inoculation screening, varieties T7, 11Q, and N13 
showed resistance-associated bands and low symptom 
severity scores compared to varieties NZ199, SC124, 
SC8 and Q12 (Figure 3). All varieties showing the 
resistance associated bands as the resistant line TME3 
were grouped under MR type. Resistance- associated 
bands were not detected in any varieties of either MS or 
HS classes. But the only variety 18R, which come under 
MS group showed same band pattern as the susceptible 
line TMS30555. Moreover, there are also some varieties 
in MR group such as R9, V5 and 16P, which come under 
the lower symptom severity scores, did not show any 



 
 
 
 

 
 

 

resistance-associated bands. 
 

 

DISCUSSION 
 

Successful agro-inoculation of cassava with 
ACMV was reported recently (Vanderschuren et 
al., 2009) and proved useful for CMD studies in 
cassava. Using agro-inoculation, 18 cassava 
varieties for CMD resistance under greenhouse 
conditions were screened. According to the 
average total symptom severity scores, cassava 
varieties were grouped into three classes: MR, MS 
and HS, respectively. In parallel, analysis of 
CMD2 associated molecular markers RME1, 
SSRY28 and NS158 showed resistance-asso-
ciated banding pattern in varieties N13, T7 and 

 

 

11Q. The result suggests that other resistance loci 
or genes related to CMD resistance might exist in 
the other cassava cultivars from the MR group.  

Most of previous screening studies for CMD 
resistance in Africa were conducted in field 
conditions under high CMD epidemic pressure 
(Akano et al., 2002; Lokko et al., 2005; Okogbenin 
et al., 2007). In this paper, agro- inoculation of 
ACMV-N Og infectious clones was used for the 
first time to screen cassava varieties from ASEAN 
countries for CMD resistance in a greenhouse. An 
agro-inoculation mediated screening system has 
several advantages compared to field screening 
studies. First, conditions for cassava growth and 
virus infection such as temperature, humidity and 
light can be controlled under greenhouse condi-
tions; this could minimize the draw-backs of field 

 

 

evaluations. Second, populations of whitefly 
vectors vary frequently with variable environ-
mental conditions such as rainfall distribution, light 
intensity and temperature (Hahn et al., 1980); 
thus, field evaluation for CMD-resistance screen-
ing that relies on vector -transmission of the virus 
can be unpredictable. Third, CMD resistance 
screening experiments in a greenhouse are safer 
than in the field in ASEAN countries, where CMD 
does not occur in cassava field yet. Finally, agro-
inoculation of cassava with ACMV infectious 
clones can facilitate the study of virus infection, 
transmission and pathogenesis.  

Nevertheless, there are some limiting factors for 

the agro-inoculation mediated screening system. 

Expression of CMD in different cassava 

genotypes is known to be dependent on the 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 3. Evaluation of cassava mosaic disease resistance in combination of agro-inoculation screening and the 
molecular marker analysis. The upper panel shows the average total symptom severity scores of cassava varieties and 

the lower panel shows the results of molecular marker analysis using RME1, SSRY28 and NS158. + means having the 
expected bands similar to the cassava mosaic disease resistant line TME3; - means no expected band detected.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Figure 4. Southern blot analysis of ACMV-NOg replication and accumulation in leaves of different cassava varieties after 

agro-inoculation. P, plasmid control; I, ACMV- NOg infected plants; M, mock inoculated control. The positions of viral 

single-stranded (ss), double-stranded (ds) and open circular (oc) DNA forms are indicated. 



  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

Figure 1. Molecular marker analysis of cassava varieties. A: SCAR marker RME1; B: SSR marker SSRY28; C: 

SSR marker NS158. M, molecular marker; 1, KU50; 2, V5; 3, GR911; 4, SC205; 5, NZ199; 6, SC8; 7, 18R; 8, SC5; 
9, N13; 10, 5E; 11, 11Q; 12, 16P; 13, 3C; 14, SC124; 15, Q12; 16, ZM9781; 17, R9; 18, T7; 19, TMS60444; 20, 

H2O; 21, TME3 (resistant line); 22, TMS30555 (susceptible line). 
 
 

 

environment, and a strong relationship between the range 

of symptoms and genotype x environment interaction has 

been reported Fargette et al., 1994). 

 
 
 

 

CMD evaluation requires evaluation tests in multiple 

environments to confirm the resistant genotypes (Lokko 

et al., 2005). Resistance screening in the green-house 



 
 
 

 

does not reflect field conditions but proves to be a rapid 
and reliable monitor system to evaluate CMD resistance 
capacity of varied cassava germplasms. In this study, all 
inoculated plants developed CMD symptoms with various 
degree of severity. One reason might be due to the lack 
of CMD resistance genes in the varieties tested as 
confirmed by the molecular marker analysis.  

After the mapping of two CMD resistance genes CMD1 
(recessive) and CMD2 (dominant) on the cassava genetic 
linkage map established by CIAT (Akano et al., 2002; 
Fregene et al., 2001), three molecular markers asso-
ciated with CMD2, namely RME1, SSRY28 and NS158, 
were developed (Lokko et al., 2007). Through cassava 
breeding programs, these markers hold great promise in 
fast tracking the CMD-resistant germplasms. For exam-
ple, with the aim to introduce Latin American germplasm 
to Africa, these markers were used to preselect neotro-
pical cassava genotypes for CMD resistance (Okogbenin 
et al., 2007). In this study, the varieties holding the CMD2 
markers are possibly from an African origin but other var-
ieties belonging to the MR group did not have the mar-
kers. One possible reason is that other genes may contri-
bute to CMD resistance besides CMD2. This is supported 
by the identification of five additional sources of resis-
tance to CMD (Legg and Fauquet, 2004).  

In this agro-inoculation screening studies, none of the 
cassava varieties tested showed immunity to virus infec-
tion but CMD resistance was associated with suppressed 
levels of viral DNA accumulation. Based upon virus accu-
mulation and symptom severity scores, cassava varieties 
were categorized into different groups. However, symp-
tom severity is not always correlated with virus concent-
ration (Ogbe et al., 2003).  

In conclusion, 18 cassava varieties from ASEAN coun-
tries were screened for CMD resistance using agro-
inoculation and molecular markers associated with the 
CMD2. This study indicated that most cassava varieties 
tested are susceptible to CMD, suggesting that those 
germpl- asms originated directly or indirectly from Latin 
America as they lacked the CMD resistant genes/ 
mechanism. It is necessary to introduce African cassava 
germplasms that harbor CMD resistance genes such as 
CMD2 to assist breeding for CMD resistance. 
 

 

ACKNOWLEDGEMENTS 
 
The authors thank Dr. John Stanley (John Innes Centre, 
U.K.) for the ACMV clones. This work was supported by 
grants from the Chinese Academy of Sciences (No. 
KSCX2-YW -G -035), the National Natural Science 
Foundation of China (No. 30771366), the National Basic 
Research Program (2010CB126605) and National High 
Technology Research and Development Program 
(2009AA10Z102) of China, the Earmarked Fund for 
Modern Agro-industry Technology Research System 
(nycytx-17) and the Shanghai Pujiang Talent Program 
(08PJ14109). 

 
 

 
 

 
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