Controlled transmission of African cassava mosaic virus ( ACMV ) by Bemisia tabaci from cassava ( Manihot esculenta Crantz ) to seedlings of physic nut ( Jatropha curcas L . )

Jatropha curcas, a plant with great biodiesel potential is also used to reduce the population of whiteflies, Bemisia tabaci on cassava fields when planted as a hedge. We therefore, investigated the transmission of African cassava mosaic virus (ACMV) by the whitefly vector from cassava to seedlings of 10 accessions of J. curcas as part of a wider investigation on the possible role of J. curcas as an alternative host of ACMV. Transmission tests were conducted in insect-proof cages using adult B. tabaci collected from ACMV-infected cassava in the field, at a rate of three adult whiteflies per J. curcas seedling and a transmission feeding period of four days. Twenty one (21) days after the infestation, leaf samples from individual plants of the 10 J. curcas accessions were tested for the presence of ACMV by the polymerase chain reaction (PCR) and the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using the monoclonal antibodies SCRI 33. DAS-ELISA detected ACMV in five out of the ten J. curcas accessions while PCR detected it in eight of the 10 accessions. Furthermore, 18 out of the 35 Nicotiana benthamiana indicator plants mechanically inoculated with sap from symptomatic J. curcas seedlings produced symptoms typical of ACMV infection. This indicates that J. curcas is a likely host of ACMV and it may in turn, be able to infect cassava, and presumably other ACMV-susceptible hosts, in the presence of the vector.


INTRODUCTION
Bemisia tabaci (Gennadius) belonging to the family Alerodidae, has been reported in all continents except Antarctica, probably due to the extremely cold conditions.The insect possibly originated in India and it is believed to have been spread throughout the world through transport of infested products (Fishpool and Burban, 1994).The insect is cosmopolitan in distribution, highly polyphagous (feeding on over 500 plant species in 74 families) and characterized by intercrop movement (Butler et al., 1986).Of the 1,100 Bemisia species identified, only three are known to be vectors of plant viruses, with B. tabaci being the most important, transmitting about 111 virus species (Brown et al., 1995) including cassava mosaic Geminiviruses (CMGs) (Legg et al., 1992).
In sub-Saharan Africa, African cassava mosaic virus (ACMV) is probably the most economically important CMG transmitted by B. tabaci (Thresh et al., 1994;Geddes, 1990).The insect has also been reported to transmit cassava brown streak virus (CBSV, family Potyviridae) which causes cassava brown streak disease (Legg et al., 1992), and Jatropha mosaic virus (Brunt et al., 1996).ACMV and CBSV have been reported to cause yield losses of cassava ranging from 20 to 90% and 70%, respectively (Gration and Rey, 2012;Hillocks et al., 2001;Thresh et al., 1994).ACMV can be controlled by using healthy in vitro materials for planting or by a hedge crop which attracts the whitefly vector, thereby reducing their populations on cassava fields.It has been demonstrated that the populations of B. tabaci in cassava fields can be reduced by planting Jatropha curcas as a hedge crop (Ewusie et al., 2010).However, recent studies have shown that ACMV can spread from infected cassava to J. curcas plants in the field (Appiah et al., 2012).Additionally, Ramkat et al. (2011) have reported the co-infection of J. curcas by ACMV and East African cassava mosaic virus-Ugandan variant (EACMV-UG) in Kenya.These recent reports of the infection of J. curcas by ACMV and EACMV-UG render the use of J. curcas as a hedge crop to trap B. tabaci inappropriate, since virus transmission from J. curcas to cassava may occur.The study reported here investigated the transmission of ACMV by B. tabaci to seedlings of J. curcas under controlled conditions, to determine the degree of susceptibility and the possible role of J. curcas as an alternative host of ACMV.

Vector transmission
Seeds of ten local accessions of J. curcas obtained from the Crop Science Department of the University of Ghana were planted in small polythene bags and arranged randomly in an insect-proof cage measuring 3 x 1.2 x 1.3 m (Figure 1).Adult B. tabaci were collected from ACMV-infected cassava (PCR and ELISA confirmed) in the field into small, fine-mesh clip cages which were placed directly onto the leaves of healthy J. curcas seedlings (Figure 2).Three adult whiteflies contained in one clip cage were placed on each J. curcas seedling and allowed a transmission access feeding period of four days, as described by Legg et al. (1992).The clip cages were then removed and the seedlings were sprayed with insecticide to kill the whiteflies and then observed for symptom expression over a period of 21 days.The percentage of viruliferous whiteflies, p, in the field collection was estimated using the formula of Gibbs and Gower (1960): Where, R is the number of infected plants, N is the total number of plants tested and i is the number of whiteflies per test plant.

Virus detection by enzyme-linked immunosorbent assay
Monoclonal antibodies (SCRI 33) for detection of ACMV were obtained from the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and used in a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), as described by Clark and Adams (1977).Three inoculated seedlings per J. curcas accession were selected at random and tested individually.An uninoculated J. curcas seedling and infected cassava were used as negative and positive controls, respectively.Leaf extract were prepared by grinding 1 g of young leaves in 10 ml of extraction buffer containing 20 g of polyvinylpyrrolidone (PVP), 2 g of ovalbumin, 1.3 g of sodium sulphite (anhydrous), 0.2 g of sodium azide, 0.5 ml of Tween, 20.8 g of sodium chloride, 0.2 g of potassium dihydrogen orthophosphate, 1.15 g of disodium hydrogen orthophosphate and 0.2 g of potassium chloride.Absorbance values were measured at 405 nm using a spectrophotometer (Multiskan Ascent VI.25-Version 1.3.1).Samples with absorbance values of more than twice that of the negative controls were considered positive for the virus.

Virus detection by polymerase chain reaction
Total DNA was extracted from fresh leaf tissues of the J. curcas seedlings according to the method described by Dellaporta et al. (1983).DNA amplification was carried out with a virus-specific primer pair JSP001 (5'-ATG TCG AAG CGA CCA GGA GAT-3')/JSP002 (5'-TGT TTA TTA ATT GCC AAT ACT-3') (Fondong et al., 2000) in a 20 µl reaction mix containing 5 µl 10x reaction buffer, 0.2 mM dNTPs, 2.5 mM MgCl 2 , 0.25 u taq polymerase, 0.2 µM forward and reverse primers and 2 µl of template DNA.Amplification reaction was performed in a 96-well Eppendorf Thermal Cycler (Eppendorf AG, Hamburg).The PCR cycle used for the amplification consisted of an initial denaturation step at 94°C for 2 min followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 58°C for 1 min and elongation at 72°C for 1 min.This was then followed by a final elongation step at 72°C for 10 min.10 µl aliquots of PCR products were mixed with 2 µl of loading dye and electrophoresed on 1% agarose gel at 90 V for 1 h.The gel was visualized under a high performance ultraviolet transilluminator (UVP, Cambridge, UK) and photographed with UVP Life Science Software (Doc -It LS Image Acquisition) and bands were scored as present or absent for analysis.

Mechanical inoculation of Nicotiana benthamiana indicator plants
Three-week old N. benthamiana indicator plants grown in an insectproof cage were used for mechanical inoculation.The N. benthamiana seeds were obtained from DSMZ Plant Virus Collection, Braunschweig, Germany.Inoculum was prepared by grinding 2 g of young leaves of J. curcas plants in 2 ml of 0.01 M phosphate buffer pH 7.0, containing sodium sulphite in a sterilized mortar and pestle.Prior to inoculation, the N. benthamiana plants were pre-conditioned by topping and removing the older lower leaves, leaving four to five leaves on a seedling.The plants were then darkened by covering with black polythene sheet overnight.The leaves were dusted with carborundum powder (600 grit) followed by rubbing the inoculum gently on the leaves with the forefinger.The inoculated leaves were then gently rinsed with tap water from a wash bottle.The inoculated plants were placed under a bench overnight and then returned to the insect-proof cage and observed for symptoms up to 21 days after inoculation Thirty five (35) N. benthamiana plants were inoculated with sap from symptomatic J. curcas seedlings, while 18 were inoculated with sap from symptomless J. curcas seedlings.Samples were collected from inoculated symptomatic plants as well as symptomless ones for ELISA detection as described previously.

ACMV transmission by B. tabaci and symptom development in J. curcas
Adult B. tabaci collected from ACMV-infected cassava in the field transmitted ACMV to seedlings of all ten accessions of J. curcas.Symptoms of leaf curling, leaf distortion and reduction in size of leaf blades, mostly of young leaves (Figure 2), were observed 21 days after the transmission feeding period.Similar symptoms have been reported in other crops as the most frequently associated with Begomovirus infection (Seal et al., 2006;Wang et al., 2004).The mosaic symptoms that normally characterizes ACMV infection of cassava (Figure 2C) were however, not evident on the infected J. curcas plants.The absence of this usual mosaic symptom on young J. curcas may be attributed to differences in the texture or the anatomy of the leaf lamina.

ELISA detection of ACMV in J. curcas
The double-antibody sandwich enzyme-linked immuno-  1).Of the 30 seedlings tested (three per accession), seven from five accessions had UV absorbance values twice that of ACMV-free, indicating that they were infected by the virus.The accession  Amanfrom, gave the highest positive absorbance of 1.006 whilst the least (0.397) was from Asamankese accession.
With three adult whiteflies per J. curcas seedling, an ACMV transmission of 23% was achieved.Our observation is comparable with that of Rashid et al. (2008) who reported 20% transmission for tomato yellow leaf curl virus (TYLCV) with three infective adult whiteflies per tomato seedling.
The low virus transmission recorded in this study and that of Rashid et al. (2008) could be attributed to the relatively low numbers of adult whiteflies per plant.Although, a single whitefly has been shown to transmit ACMV, the percent transmission increased with increaseing number of whiteflies per plant (Mahto and Sinha, 1978).Mathew (1988) achieved 100% transmit-ssion of Indian cassava mosaic virus to N. benthamiana with over 40 adult whiteflies per plant.Similarly, Nguessan et al. (1992) achieved 50% transmission of okra leaf curl virus (OLCV) when each plant was infested with 25 adult whiteflies, even though the estimated percentage of viruliferous whiteflies was 4% as compared to the 8.5% recorded in this study.

Polymerase chain reaction detection of ACMV in J. curcas
The polymerase chain reaction detected ACMV in all the J. curcas accessions except Gbefi and Apeguso (Figure 3).The technique was able to detect the virus in accessions Valley View University, Aklamado and Kpeve that tested negative to DAS-ELISA.This could be due to the enhanced sensitivity of the PCR technique (Martin, 1998) as compared to ELISA, making it the preferred method for the detection and characterization of viral infections that are difficult to detect and diagnose by serology (Seal and Coates, 1998).Disparities in the detection of ACMV by ELISA and PCR have been reported by Okorogri et al. (2010).
The primer pair JSP001/JSP002 amplified the expected products of size ~0.7 kb in the coat protein gene of the virus.Adjata et al. (2008a), Mbasa (2007) and Pita et al. (2001) have independently used this same primer for successful detection of ACMV in cassava.

Figure 1 .
Figure 1.A, Insect-proof cage (3 x 1.2 x 1.3 m) with J. curcas seedlings.B, clip cage containing trapped B. tabaci and attached to a leaf of Jatropha seedling to facilitate access feeding.

Figure 2 .
Figure 2. A, Whitefly-inoculated J. curcas plant showing curling and distortion of young leaves.B, Healthy uninoculated J. curcas plant, C, Field infected cassava showing severe symptoms of ACMD.

Table 1 .
DAS-ELISA detection of African cassava mosaic virus in leaf extracts of ten accessions of J. curcas seedlings inoculated by adult Bemisia tabaci.