- Email: [email protected]
Assessing aphids potato virus Y-transmission efficiency: A new approach
Journal of Virological Methods 178 (2011) 63–67
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Assessing aphids potato virus Y-transmission efficiency: A new approach Sébastien Boquel a,b,∗ , Arnaud Ameline b , Philippe Giordanengo b a b
GIE, Station de Recherche et de Création Variétale du Comité Nord, 76110 Bretteville-du-Grand-Caux, France Université de Picardie Jules Verne, Laboratoire de Biologie des Entomophages, 33 rue Saint Leu, 80039 Amiens Cedex, France
a b s t r a c t Article history: Received 29 April 2011 Received in revised form 8 August 2011 Accepted 14 August 2011 Available online 22 August 2011 Keywords: Solanum tuberosum In vitro plantlets Electrical penetration graph (EPG) Virus transmission Aphids
In order to develop an alternative method to optimize the relative efficiency factor (REF) assessment, the efficiency of transmission of Potato virus Y (PVY) by seven aphid species was examined. In vitro micropropagated potato plantlets were used to experiment on phenotypically and genetically homogeneous material. Species-specific acquisition access period (AAP) on a PVY-infected plantlet was assessed for each aphid species using electrical penetration graph (EPG) technique. Aphid probing behaviour determined by EPG showed that Macrosiphum euphorbiae and Myzus persicae exhibited the shortest AAPs (15 and 11 min, respectively) whereas Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae and Acyrthosiphon pisum exhibited the longest ones (more than 30 min). The transmission rate obtained for M. persicae (83.3%) was higher than the ones reported in the literature. REFs assessment showed that A. pisum and B. brassicae were poor efficient vectors while M. euphorbiae and S. avenae seemed to be efficient ones even though their respective REF were significantly lower than that of M. persicae. The species R. padi and A. fabae did not transmit PVY. The hypothesis assessed for M. euphorbiae and S. avenae and consisting in the compensation of a weak PVY-transmission efficiency by a higher number of vectors, was not supported. The use of this new method for REF evaluation and the need to consider aphid behaviour for such an assessment was discussed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Potato virus Y (PVY, Potyviridae: Potyvirus) has a huge economic impact on the potato (Solanum tuberosum L.) production system (Sigvald, 1992) as it can cause tuber necrosis leading to unmarketable harvest. Under field conditions, PVY is transmitted efficiently in a non-persistent manner by potato colonizing aphids such as the green peach aphid Myzus persicae (Sulzer) and the potato aphid Macrosiphum euphorbiae (Thomas) (Radcliffe and Ragsdale, 2002). Some non-colonizing potato aphid species have also been reported to transmit PVY despite lower transmission rates. Field samples showed high virus infection rates in potato plots even when very low densities of potato colonizing aphids were reported (Boiteau et al., 1998) and it has been suggested that in the absence of potato colonizing species the massively trapped
Abbreviations: AAP, acquisition access period; ELISA, enzyme-linked immunosorbent assay; EPG, electrical penetration graph; IAP, inoculation access period; MS, Murashige and Skoog; PCR, polymerase chain reaction; PVY, potato virus Y; REF, relative efficiency factor; RT, reverse transcription. ∗ Corresponding author. Tel.: +33 322 827 547; fax: +33 322 827 547. E-mail addresses: [email protected] (S. Boquel), [email protected] (A. Ameline), [email protected] (P. Giordanengo). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.08.013
non-colonizing aphids may be responsible for the spread of PVY (DiFonzo et al., 1997). Many studies measuring and estimating PVY vector efficiency revealed intraspecific variations (Harrington and Gibson, 1989; Sigvald, 1984; van Harten, 1983; van Hoof, 1980; Verbeek et al., 2010). However, all of the investigations agreed to consider M. persicae as the most efficient PVY vector and used it as a reference. The transmission efficiency of other aphid vectors is therefore expressed as a relative efficiency factor (REF) related to that of M. persicae set to 1 (van Harten, 1983). Within an aphid species, the variability of REF reported in the literature can result from the aphid biotype and the number of aphids used to infect plants experimentally (Halbert et al., 2003; Lupoli et al., 1992; Moreno et al., 2007; Verbeek et al., 2010), the PVY strains and isolates (Verbeek et al., 2010), the target plant species and cultivar (Fereres et al., 1993; Hamm et al., 2010; Singh and Boiteau, 1986), the plant phenological state (Woodford, 1992) and the virus concentration in the plant source (Pirone and Megahed, 1966). Finally, REF variability can rely on the aphid probing behaviour as efficient acquisition and inoculation are realised during brief intracellular punctures (3–6 s) performed during the first steps of host plant colonisation process (Powell et al., 2006). Therefore, successful vectoring depends firstly on the acquisition access period (AAP) that corresponds to the time elapsed from the aphid contact with the infected plant to the first intracellular puncture.
64
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
Basically, two methods have been used to assess vector efficiency. First, aphids are placed in a closed arena containing both infected and healthy plants (Kanavaki et al., 2006; Katis et al., 2006). However, such a set-up does not allow us to distinguish the contribution of aphid behaviour in the transmission process (vector activity) from their intrinsic ability to transmit viruses (vector efficiency). The second experimental approach aims to control the transmission process by placing the aphid vector on an infected plant for an AAP, and then transferring it to a healthy plant for an inoculation access period (IAP). While the latter method minimizes vector activity contribution as the vectors are artificially placed on the source and then on the target plant, they are usually performed with short AAPs that can induce bias in the vector efficiency assessment. Indeed, it has been demonstrated that AAP varies depending on the aphid species and is modulated by the healthy or infected status of the plant (Boquel et al., 2011). The main objective of this paper was to develop, on the aphid vector – PVY – potato pathosystem, an alternative method to optimize REF assessment by minimizing the part of the source and target plants and the part of vector. In vitro micropropagated potato plantlets were used to get phenotypically and genetically homogeneous plant material (Acquaah, 2007; Hu and Wang, 1983) and minimize potato plant defence responses (Péros et al., 1994; Petroviˇc et al., 1997), and clones of seven aphid species (M. euphorbiae, M. persicae, Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae, Aphis fabae and Acyrthosiphon pisum) were used to minimize intraspecific variability. To ensure the vector a sufficient contact time with the plant to perform intracellular puncture and acquire virus particles, electrical penetration graph (EPG) technique was used to define a specific AAP for each aphid species. Finally, this method was used to test the hypothesis suggested by DiFonzo et al. (1997) who postulated that a weak vector efficiency could be compensated by high vector densities in fields.
(INRA-INSA-CIRAD, Montpellier). Each aphid clone, started from a virus free single apterous parthenogenetic female, was reared on a healthy host plant enclosed in a ventilated Plexiglas® cage (360 mm × 240 mm × 110 mm) in a growth chamber (20 ± 1 ◦ C, 60 ± 5% relative humidity, 16:8 h day:night cycle). M. persicae and M. euphorbiae were reared on potato (S. tuberosum L.), A. fabae and A. pisum on broad bean (Vicia faba L.), S. avenae and R. padi on wheat (Triticum aestivum L.) and B. brassicae on broccoli (Brassica oleracea L.). All experiments were performed at 20 ± 1 ◦ C with alates synchronized in host plant seeking phase according to the set-up described by Brunissen et al. (2009).
2.3. Evaluation of a species-specific acquisition access period (AAP) The DC-electrical penetration graph (EPG) technique (Tjallingii, 1978, 1988) was used to determine the necessary time for each aphid species to perform the first potential drop (intracellular puncture) on a PVY-infected in vitro potato plantlet. To insert one aphid and one plant in an electrical circuit, a thin gold wire (20 m diameter, 2 cm long) was stuck with conductive water based silver glue (EPG-systems, Wageningen, The Netherlands) on the aphid’s dorsum and a second electrode was inserted into the agar-based MS medium. The aphid was then connected to the DC-EPG amplifier and placed carefully on a PVY-infected in vitro plantlet into a Faraday cage at an ambient temperature of 20 ± 1 ◦ C. For each aphid species 20 replicates were done. The recordings were performed during daytime for 8 continuous hours. Acquisition and analysis of the EPG waveforms were done with PROBE 3.5 software (EPG-Systems, Wageningen, The Netherlands) and AAP, defined as the mean time from start of the monitoring (when the aphid was deposited on the leaflet) to the first potential drop added to its standard error, was calculated using the EPG-Calc 4.9 software (Giordanengo, 2009).
2. Materials and methods 2.1. Virus isolate and in vitro plantlets The PVYNTN isolate was obtained from potato tubers collected in a potato field (Cambrai, France). Healthy and PVYNTN -infected in vitro potato (cv. Bintje) lines were obtained from germ fragments (2–3 cm), respectively, collected from a healthy and an infected tuber, washed during 20 min with a 7% calcium hypochlorite solution, dried with blotting paper and deposited in a Murashige and Skoog (1962) (MS basal medium with sucrose and agar, 42.4 g L−1 ) medium for development. For micropropagation, explants were isolated on MS medium in a small glass vial (5 mL) placed in a sterile culture glass tube (25 mm × 150 mm) in a growth chamber at 20 ± 1 ◦ C, 60 ± 5% relative humidity and 16:8 h day:night cycle. Each small glass vial containing one 15 day-old healthy or PVYinfected in vitro plantlet was withdrawn from the glass tube for experiments. Immunocapture RT-PCR technique was used to determine the healthy or infected status of the plantlet according to Glais et al. (1998). 2.2. Aphids Aphid species used for virus transmission bioassays were chosen according to their abundance in potato fields in northern France or their ability to transmit PVY (Harrington and Gibson, 1989; Sigvald, 1984; van Harten, 1983; van Hoof, 1980). Aphids were obtained from different French localities or laboratories: M. persicae (potato field, Loos-en-Gohelle), M. euphorbiae (INRAINSA, Villeurbanne), A. fabae (eggplant greenhouse, Amiens), S. avenae and R. padi (INRA, Le Rheu), A. pisum and B. brassicae
2.4. Transmission experiments and relative efficiency factors (REFs) evaluation For each aphid species, 5 synchronized alates were deposited on a PVY-infected in vitro potato plantlet placed in a Plexiglas® box (120 mm × 90 mm × 50 mm) during a period equal to their specific AAP. Then, aphids were deposited individually on a healthy in vitro plantlet grown in the culture glass tube for a 24 h IAP. Finally, after IAP, aphids and their possible progeny were removed and in vitro plantlets were placed in a growth chamber for a 15-day-incubation period before checking for PVY infection by immunocapture RTPCR. For each aphid species, 30 individuals were tested. The REF of each aphid species was calculated by dividing the number of infected plants obtained with the considered species by the number of infected plants obtained with M. persicae used as referent species.
2.5. Number vs efficiency DiFonzo et al. (1997) hypothesized that the high numbers of non-colonizing aphids could compensate their weak virus transmission efficiency. In accordance to the obtained results in transmission experiments, this hypothesis was tested with the two aphid species that ranked, respectively, second and third in the calculated REF. The number of potentially viruliferous aphids deposited simultaneously per target plantlet for IAP was calculated according to the equation: number aphid to use = REF M. persicae/REF tested species. Then, transmission efficiency experiments were realised according to the method described above.
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
65
Table 1 Species-specific acquisition access period (AAP) determined for seven aphid species with electrical penetration graph technique during an-8-h recording on PVY infected in vitro S. tuberosum cv. Bintje plantlets.
Fpd ± SE (min) AAP (min)
M. euphorbiae
M. persicae
A. fabae
R. padi
A. pisum
S. avenae
B. brassicae
12 ± 3 ab 15
9±2 a 11
72 ± 18 d 90
25 ± 6 cd 31
20 ± 10 abc 30
28 ± 7 bcd 35
37 ± 11 cd 48
Different letters indicate significant difference at p < 0.05 according to a Mann–Whitney test after a Kruskal–Walllis test. Fpd, time from start of the recording to the first intracellular puncture (i.e. potential drop); SE, standard error.
2.6. Data analysis Statistical analyses were performed using STATISTICA 8.0 software (StatSoft, Tulsa, OK, USA). Transmission rates were calculated as percentages of infected plants and were compared with the different aphid species using a Chi-square test. Because EPG data were not normally distributed, pairwise comparisons were done between the different aphid species with a Mann–Whitney U-test after a global analyse with the Kruskal–Wallis (H) test. Significant differences were determined at p < 0.05. 3. Results 3.1. Evaluation of species-specific acquisition access period (AAP) For each aphid species, different AAP were obtained (Table 1). M. persicae showed the shortest AAP (11 min) followed by M. euphorbiae (15 min). AAP of non-colonizing aphid species varied from 30 min for A. pisum to 48 min for B. brassicae except for A. fabae which took 90 min to realise the first potential drop. 3.2. Transmission experiments and relative efficiency factors (REFs) evaluation Most aphid species tested transmitted PVYNTN isolate with different transmission rates. M. persicae exhibited the greatest rate of transmission with 83.3% and its REF was set to 1 (Table 2). The other potato colonizing aphid M. euphorbiae exhibited a significant lower transmission rate (26.7%, REF = 0.32, 2 = 17.24, p < 0.01) than that of M. persicae. Among non-colonizing aphids species, S. avenae and A. pisum showed intermediate transmission rates (16.7%, REF = 0.20 and 6.7%, REF = 0.08, respectively), which only significantly differed from that of M. persicae (2 = 24.07, p < 0.01; 2 = 32.59, p < 0.01, respectively). The three remaining species, B. brassicae (3.3%, REF = 0.04), A. fabae (0%, REF = 0.00) and R. padi (0%, REF = 0.00), exhibited significant lower transmission rates compared to that of both potato colonizing aphids M. persicae (2 = 35.90, p < 0.01; 2 = 39.50, p < 0.01; 2 = 39.50, p < 0.01, respectively) and M. euphorbiae (2 = 4.71, p < 0.05; 2 = 7.07, p < 0.01; 2 = 7.07, p < 0.01, respectively).
Table 2 PVY transmission efficiency and relative efficiency factors (REFs) of seven aphid species. Aphid species
Number of aphid per plant
Infected plantlets (%)
REF
M. persicae M. euphorbiae A. pisum A. fabae B. brassicae R. padi S. avenae
1 1 1 1 1 1 1
83.3 a 26.7 b 6.7 bc 0c 3.3 c 0c 16.7 bc
1.00 0.32 0.08 0.00 0.04 0.00 0.20
Different letters indicate significant difference between species at p < 0.05 according to a Chi-square test. REFs were calculated according to the equations describe in Materials and methods section (2.4).
Table 3 PVY transmission efficiency and relative efficiency factors (REFs) of 3 aphid species according to the number of aphids used to infect target in vitro S. tuberosum cv. Bintje plantlets. Aphid species
Number of aphid per plant
Infected plantlets (%)
REF
M. persicae M. euphorbiae S. avenae
1 3 5
83.3 a 3.3 b 23.3 b
1.00 0.04 0.28
Different letters indicate significant difference between species at p < 0.05 according to a Chi-square test. Number of aphid per plant and REFs were calculated according to the equations describe in Materials and methods section (2.5 and 2.4, respectively).
3.3. Number vs efficiency As M. euphorbiae and S. avenae ranked respectively second and third in REF, the number of potentially viruliferous aphids to use for IAP was calculated for both species. It was nearly equal to 3 for M. euphorbiae and 5 for S. avenae. Results of transmission efficiencies showed that for neither of the two species (M. euphorbiae: 3.3%, 2 = 35.90, p < 0.01 and S. avenae: 23.3%, 2 = 19.35, p < 0.01) did a higher number of vectors compensate their weak transmission rate compared to M. persicae (Table 3). Surprisingly, increasing the number of viruliferous aphids on a target plantlet during the IAP led to reduced transmission efficiency for M. euphorbiae (3.3% vs 26.7%, 2 = 4.71, p < 0.05) and an unchanged transmission efficiency for S. avenae (23.3% vs 16.7%, 2 = 0.10, p = 0.75). 4. Discussion With the objective to evaluate the vector efficiency of aphid species, REF values can be estimated reliably by assessing transmission rates of individual aphids using species-specific AAPs and in vitro plantlets as inoculum source and target plants. The transmission rate obtained for M. persicae (83.3%) was higher than the one reported in the literature irrespective of the M. persicae clone used (Halbert et al., 2003; Sigvald, 1984; Verbeek et al., 2010). It was also higher than the one obtained previously for the same M. persicae clone (45%) on plants grown from tubers from the same S. tuberosum cultivar Bintje (data not shown). Such discrepancy observed in transmission efficiency could be attributed to the plant material used (in vitro vs tuber-grown potato plants) rather than to the aphid clone used. Compared to tuber-grown plants, in vitro plantlets may be unable to develop efficient defence responses towards pathogens (Péros et al., 1994; Petroviˇc et al., 1997) thus leading to successful virus infection. Consequently, one could expect higher virus concentration in in vitro material as reported by Petroviˇc et al. (1997) and a more homogeneous distribution of viral particles within plantlet tissues. Moreover, as confirmed by the semiquantitative RT-PCR performed on in vitro potato plantlets used as source for AAP in the transmission experiments, virus concentrations were constant and no significant difference was measured between plantlet series (data not shown). Finally, the RT-PCR technique used in this study allows a more accurate assessment of the infected status of the plant as the detection threshold of viral particles is lower than the commonly used ELISA technique (Moreno et al., 2007; Wang and Ghabrial, 2002).
66
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
The EPG monitoring revealed that the time elapsed from the aphid contact with the infected plant until the first intracellular puncture (AAP), necessary for virus acquisition (Powell et al., 1995), differed between aphid species. M. persicae and M. euphorbiae took less than 15 min to achieve the first intracellular puncture while non-colonizing aphid species needed more than 30 min to exhibit such behaviour. These different AAPs between colonizing and non-colonizing aphids could result from their host plant range. M. persicae and M. euphorbiae are polyphagous aphids and S. tuberosum is one of their favoured secondary hosts (Blackman and Eastop, 2000). By contrast, as potato is not a host plant for the non-colonizing aphid species (Blackman and Eastop, 2000), delayed times in their first intracellular puncture could be associated to a lower plant acceptance. The variations in AAPs observed could also be due, at least partly, to the infected status of the plant. It is assumed that plant infection by phytoviruses can affect behaviour and physiology of vectoring aphids (Fereres and Moreno, 2009). On PVY-infected potato plantlets, M. persicae exhibited increased phloem ingestion and reduced non-probing duration whereas M. euphorbiae showed delayed stylet insertion, reduced activity in the phloem vessels and an enhanced nonprobing duration (Boquel et al., 2011). These results suggested that these behavioural variations might be a consequence of a physical modification or the induction of chemical compounds (e.g. antifeedant/phagostimulant) in vascular and mesophyll tissues. Such results underline that it is crucial to measure species-specific AAP in order to reliably evaluate vector efficiency. In most of the published data, a single AAP of generally 5 min after proboscis contact was used whatever the aphid species (Fernández-Calvino et al., 2006; Katis et al., 2006; Verbeek et al., 2010) leading to underestimated REFs. Indeed, proboscis contact with the plant did not revealed an efficient acquisition, as intracellular puncture was not highlighted. In this work, a method using the EPG tool that allows estimation of a more accurate AAP was proposed. These experiments confirmed M. persicae as the most efficient vector of PVY. M. euphorbiae appeared as an efficient vector with 26.7% transmission rate whereas it was reported as a weak or a non vector of PVY (Harrington and Gibson, 1989; Singh and Boiteau, 1986; Verbeek et al., 2010). Whatever the clone tested, no or very low transmission rates were observed with S. avenae (de Bokx and Piron, 1990; Piron, 1986; van Hoof, 1980; Verbeek et al., 2010). Surprisingly, a high PVY REF was obtained for S. avenae clone, suggesting its significant involvement in the spread of PVY. In accordance with the literature, R. padi, A. fabae, B. brassicae and A. pisum showed very low or even undetectable transmission rates (de Bokx and Piron, 1990; Harrington and Gibson, 1989; Heimbach et al., 1998; Piron, 1986; Verbeek et al., 2010). DiFonzo et al. (1997) suggested that aphids showing weak vector efficiencies might play an important role in the epidemics of non-persistent viruses provided that their field population densities are high. In these experimental conditions, increasing the number of viruliferous aphids deposited on the target plant did not enhance the transmission efficiencies and then did not corroborate this hypothesis. The REF of S. avenae was unchanged by increasing the number of viruliferous aphids set on a plant. Surprisingly, increasing the number of M. euphorbiae set on a healthy plant during the IAP decreased its vector efficiency. M. euphorbiae is a fairly mobile species (Boiteau, 1997) and did not settle easily on the small in vitro plantlets (pers. obs.) which could reduce virus transmission. Moreover, a high aphid number could enhance the plantlet defence responses as reported on tuber-grown potato plants (Ameline et al., 2007; Dugravot et al., 2007). This could explain, at least partly, the low efficiency rates reported in the previous works where up to 5 aphids were used per target plant (Kalleshwaraswamy and Krishna-Kumar, 2007; Katis et al., 2006; van Hoof, 1980). In a methodological context, using only one aphid
as a vector on the target in vitro plantlet seems to be sufficient to estimate the REF and would avoid bias consequent to intraspecific interactions. However, because of the limitations imposed by the in vitro model preventing the use of numerous aphids, the DiFonzo et al. (1997) hypothesis was not tested with either aphids exhibiting low vector efficiencies or with higher numbers of M. euphorbiae or S. avenae. Brought together with the high viral infection rates even in the absence of colonizing aphids which exhibit higher transmission efficiencies, these results cannot lead to preclude that successful spread of PVY in the fields could result from the massive densities of non colonizing aphids. Therefore, it would be worth including other parameters such as interplant movements to validate this hypothesis since non-colonizing aphid species are more restless than colonizing ones (Nemecek et al., 1993). To conclude, a reliable method to evaluate PVY transmission efficiency is proposed. Species-specific AAPs evaluation insures optimal virus acquisition and using in vitro plants allows saving time and space regarding their rapid growth and small size. With the aim to develop a reliable method to assess REFs, aphid material was standardized by using only one clone. As variations in transmission rates can occur between different clones (Woodford, 1992), epidemic studies would integrate such intraspecific variations and compare the vector efficiency of several clones to improve REF assessment of the considered species.
Acknowledgements This work was funded by the Conseil Régional de Picardie and the Comité Nord Plants de Pommes de Terre. We thank D. Tagu (INRA, Rennes, France) for providing the S. avenae and R. padi clones, M. Uzest (INRA-CIRAD, Montpellier, France) for A. pisum and B. brassicae clones, Y. Rahbé (INRA-INSA, Villeurbanne) for M. euphorbiae clone and F. Lemoine and X. Riquiez from the Comité Nord Plants de Pommes de Terre for providing healthy in vitro plant lineage and infected tuber. The authors also thank Aude Couty, Andrew Roots and Mamed Ferkioui for their advice in the usage of the English language.
References Acquaah, G., 2007. Principles of Plant Genetics and Breeding. Blackwell Publishing, Oxford. Ameline, A., Couty, A., Dugravot, S., Campan, E., Dubois, F., Giordanengo, P., 2007. Short-term alteration of Macrosiphum euphorbiae host plant selection behaviour after biotic and abiotic damages inflicted to potato plants. Entomol. Exp. Appl. 123, 129–137. Blackman, R.L., Eastop, V.F., 2000. Aphids on the World’s Crop: An Identification and Information Guide, Second ed. John Wiley & Soons LTD, New York. Boiteau, G., 1997. Comparative propensity for dispersal of apterous and alate morphs of three potato-colonizing aphid species. Can. J. Zool. 75, 1396–1403. Boiteau, G., Singh, M., Singh, R.P., Tai, G., Turner, T., 1998. Rate of spread of PVYN by alate Myzus persicae (Sulzer) from infected to healthy plants under laboratory conditions. Potato Res. 41, 335–344. Boquel, S., Giordanengo, P., Ameline, A., 2011. Divergent effects of PVY-infected potato plant on aphid. Eur. J. Plant Pathol. 129, 507–510. Brunissen, L., Cherqui, A., Pelletier, Y., Vincent, C., Giordanengo, P., 2009. Host–plant mediated interactions between two aphid species. Entomol. Exp. Appl. 132, 30–38. de Bokx, J.A., Piron, P.G.M., 1990. Relative efficiency of a number of aphid species in the transmission of Potato virus YN in the Netherlands. Neth. J. Plant Pathol. 96, 237–246. DiFonzo, C.D., Ragsdale, D.W., Radcliffe, E.B., Gudmestad, N.C., Secor, G.A., 1997. Seasonal abundance of aphid vectors of Potato virus Y in the Red River Valley of Minnesota and North Dakota. J. Econ. Entomol. 90, 824–831. Dugravot, S., Brunissen, L., Letocart, E., Tjallingii, W.F., Vincent, C., Giordanengo, P., Cherqui, A., 2007. Local and systemic responses induced by aphids in Solanum tuberosum plants. Entomol. Exp. Appl. 123, 271–277. Fereres, A., Moreno, A., 2009. Behavioural aspects influencing plant virus transmission by homopteran insects. Virus Res. 141, 158–168. Fereres, A., Perez, P., Gemeno, C., Ponz, F., 1993. Transmission of Spanish pepper- and potato-PVY isolates by aphid (Homoptera: Aphididae) vector: epidemiological implications. Environ. Entomol. 22, 1260–1265.
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67 Fernández-Calvino, L., López-Abella, D., López-Moya, J.J., Fereres, A., 2006. Comparison of Potato virus Y and Plum pox virus transmission by two aphid species in relation to their probing behavior. Phytoparasitica 34, 315–324. Giordanengo, P., 2009. EPG-Calc: A PHP Program to Evaluate Epg Parameters. Université de Picardie Jules Verne, Available online: http://www.upicardie.fr/PCP/UTIL/epg.php. Glais, L., Tribodet, M., Gauthier, J.P., Astier-Manifacier, S., Robaglia, C., Kerlan, C., 1998. RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of Potato virus Y. Arch. Virol. 143, 2077–2091. Halbert, S.E., Corsini, D.L., Wiebe, M.A., 2003. Potato virus Y transmission efficiency for some common aphids in Idaho. Am. J. Potato Res. 80, 87–91. Hamm, P.B., Hane, D.C., Pavek, M.J., Leroux, L.D., Gieck, S.L., David, N.L., 2010. Potato cultivars differ in current season Potato Virus Y (PVY) infection. Am. J. Potato Res. 87, 19–26. Harrington, R., Gibson, R.W., 1989. Transmission of Potato virus Y by aphids trapped in potato crop in southern England. Potato Res. 32, 167–174. Heimbach, U., Thieme, T., Weidemann, H.L., Thieme, R., 1998. Transmission of Potato virus Y by aphid species which do not colonise potatoes. In: Nieto-Nafria, J.M., Dixon, A.F.G. (Eds.), Aphids in natural and managed ecosystems. Universitad de León, León, pp. 555–559. Hu, C.Y., Wang, P.J., 1983. Meristem shoot tip and bud cultures. In: Evans, D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. 1. MacMillan Publishing Company, New York, pp. 177–227. Kalleshwaraswamy, C.M., Krishna-Kumar, N.K., 2007. Transmission efficiency of Papaya ringspot virus by three aphid species. Phytopathology 98, 541–546. Kanavaki, O.M., Margaritopoulos, J.T., Katis, N.I., 2006. Transmission of Potato virus Y in tobacco plants by Myzus persicae nicotianae and M. persicae s.str. Plant Dis. 90, 777–782. Katis, N.I., Tsitsipis, J.A., Lykouressis, D.P., Papapanayotou, A., Margaritopoulos, J.T., Kokinis, G.M., Perdikis, D.C., Manoussopoulos, I.N., 2006. Transmission of Zucchini yellow mosaic virus by colonizing and non-colonizing aphids in Greece and new aphid species vectors of the virus. J. Phytopathol. 154, 293–302. Lupoli, R., Labonne, G., Yvon, M., 1992. Variability in the transmission efficiency of potyviruses by different clones of Aphis gossypii. Entomol. Exp. Appl. 65, 291–300. Moreno, A., Bertolini, E., Olmos, A., Cambra, M., Fereres, A., 2007. Estimation of vector propensity for Lettuce mosaic virus based on viral detection in single aphids. Span. J. Agric. Res. 5, 376–384. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.
67
Nemecek, T., Fischlin, A., Derron, J., Roth, O., 1993. Quantifying behaviour sequences of winged aphids on potato plants for virus epidemic models. Bull. Entomol. Res., 1–16. Péros, J.P., Bonnel, E., Roques, D., Paulet, F., 1994. Effect of in vitro culture on rust resistance and yield in sugarcane. Field Crops Res. 37, 113–119. Petroviˇc, N., Miersch, O., Ravnikar, M., Kovaˇc, M., 1997. Potato virus YNTN alters the distribution and concentration of endogenous jasmonic acid in potato plants grown in vitro. Physiol. Mol. Plant Pathol. 50, 237–244. Piron, P.G.M., 1986. New aphid vectors of Potato virus YN . Neth. J. Plant Pathol. 92, 223–229. Pirone, T.P., Megahed, E.S., 1966. Aphid transmissibility of some purified viruses and viral RNAs. Virology 30, 631–637. Powell, G., Pirone, T.P., Hardie, J., 1995. Aphid stylet activities during potyvirus acquisition from plants and an in vitro system that correlate with subsequent transmission. Eur. J. Plant Pathol. 101, 411–420. Powell, G., Tosh, C.R., Hardie, J., 2006. Host plant selection by aphids: behavioral, evolutionary, and applied perspectives. Annu. Rev. Entomol. 51, 309–330. Radcliffe, E.B., Ragsdale, D.W., 2002. Aphid-transmitted potato viruses: the importance of understanding vector biology. Am. J. Potato Res. 79, 353–386. Sigvald, R., 1984. The relative efficiency of some aphid species as vectors of Potato virus YO (PVYO ). Potato Res. 27, 285–290. Sigvald, R., 1992. Progress in aphid forecasting systems. Neth. J. Plant Pathol. 98, 55–62. Singh, R.P., Boiteau, G., 1986. Reevaluation of the potato aphid, Macrosiphum euphorbiae (Thomas), as vector of Potato virus Y. Am. J. Potato Res. 63, 335–340. Tjallingii, W.F., 1978. Electronic recording of penetration behaviour by aphids. Entomol. Exp. Appl. 24, 521–530. Tjallingii, W.F., 1988. Electrical recording of stylet penetration activities. In: Minks, A.K., Harrewijn, P. (Eds.), Aphids: Their Biology, Natural Enemies and Control, vol. 2B. Elsevier, Amsterdam, pp. 95–108. van Harten, A., 1983. The relation between aphid flights and the spread of Potato virus YN (PVYN ) in the Netherlands. Potato Res. 26, 1–15. van Hoof, H.A., 1980. Aphid vectors of Potato virus YN . Neth. J. Plant Pathol. 86, 159–162. Verbeek, M., Piron, P.G.M., Dullemans, A.M., Cuperus, C., van der Vlugt, R.A.A., 2010. Determination of aphid transmission efficiencies for N. NTN and Wilga strains of Potato virus Y. Ann. Appl. Biol. 156, 39–49. Wang, R.Y., Ghabrial, S.A., 2002. Effect of aphid behavior on efficiency of transmission of Soybean mosaic virus by the soybean-colonizing aphid, Aphis glycines. Plant Dis. 86, 1260–1264. Woodford, J.A.T., 1992. Virus transmission by aphids in potato crops. Neth. J. Plant Pathol. 2, 47–54.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Assessing aphids potato virus Y-transmission efficiency: A new approach Sébastien Boquel a,b,∗ , Arnaud Ameline b , Philippe Giordanengo b a b
GIE, Station de Recherche et de Création Variétale du Comité Nord, 76110 Bretteville-du-Grand-Caux, France Université de Picardie Jules Verne, Laboratoire de Biologie des Entomophages, 33 rue Saint Leu, 80039 Amiens Cedex, France
a b s t r a c t Article history: Received 29 April 2011 Received in revised form 8 August 2011 Accepted 14 August 2011 Available online 22 August 2011 Keywords: Solanum tuberosum In vitro plantlets Electrical penetration graph (EPG) Virus transmission Aphids
In order to develop an alternative method to optimize the relative efficiency factor (REF) assessment, the efficiency of transmission of Potato virus Y (PVY) by seven aphid species was examined. In vitro micropropagated potato plantlets were used to experiment on phenotypically and genetically homogeneous material. Species-specific acquisition access period (AAP) on a PVY-infected plantlet was assessed for each aphid species using electrical penetration graph (EPG) technique. Aphid probing behaviour determined by EPG showed that Macrosiphum euphorbiae and Myzus persicae exhibited the shortest AAPs (15 and 11 min, respectively) whereas Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae and Acyrthosiphon pisum exhibited the longest ones (more than 30 min). The transmission rate obtained for M. persicae (83.3%) was higher than the ones reported in the literature. REFs assessment showed that A. pisum and B. brassicae were poor efficient vectors while M. euphorbiae and S. avenae seemed to be efficient ones even though their respective REF were significantly lower than that of M. persicae. The species R. padi and A. fabae did not transmit PVY. The hypothesis assessed for M. euphorbiae and S. avenae and consisting in the compensation of a weak PVY-transmission efficiency by a higher number of vectors, was not supported. The use of this new method for REF evaluation and the need to consider aphid behaviour for such an assessment was discussed. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Potato virus Y (PVY, Potyviridae: Potyvirus) has a huge economic impact on the potato (Solanum tuberosum L.) production system (Sigvald, 1992) as it can cause tuber necrosis leading to unmarketable harvest. Under field conditions, PVY is transmitted efficiently in a non-persistent manner by potato colonizing aphids such as the green peach aphid Myzus persicae (Sulzer) and the potato aphid Macrosiphum euphorbiae (Thomas) (Radcliffe and Ragsdale, 2002). Some non-colonizing potato aphid species have also been reported to transmit PVY despite lower transmission rates. Field samples showed high virus infection rates in potato plots even when very low densities of potato colonizing aphids were reported (Boiteau et al., 1998) and it has been suggested that in the absence of potato colonizing species the massively trapped
Abbreviations: AAP, acquisition access period; ELISA, enzyme-linked immunosorbent assay; EPG, electrical penetration graph; IAP, inoculation access period; MS, Murashige and Skoog; PCR, polymerase chain reaction; PVY, potato virus Y; REF, relative efficiency factor; RT, reverse transcription. ∗ Corresponding author. Tel.: +33 322 827 547; fax: +33 322 827 547. E-mail addresses: [email protected] (S. Boquel), [email protected] (A. Ameline), [email protected] (P. Giordanengo). 0166-0934/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2011.08.013
non-colonizing aphids may be responsible for the spread of PVY (DiFonzo et al., 1997). Many studies measuring and estimating PVY vector efficiency revealed intraspecific variations (Harrington and Gibson, 1989; Sigvald, 1984; van Harten, 1983; van Hoof, 1980; Verbeek et al., 2010). However, all of the investigations agreed to consider M. persicae as the most efficient PVY vector and used it as a reference. The transmission efficiency of other aphid vectors is therefore expressed as a relative efficiency factor (REF) related to that of M. persicae set to 1 (van Harten, 1983). Within an aphid species, the variability of REF reported in the literature can result from the aphid biotype and the number of aphids used to infect plants experimentally (Halbert et al., 2003; Lupoli et al., 1992; Moreno et al., 2007; Verbeek et al., 2010), the PVY strains and isolates (Verbeek et al., 2010), the target plant species and cultivar (Fereres et al., 1993; Hamm et al., 2010; Singh and Boiteau, 1986), the plant phenological state (Woodford, 1992) and the virus concentration in the plant source (Pirone and Megahed, 1966). Finally, REF variability can rely on the aphid probing behaviour as efficient acquisition and inoculation are realised during brief intracellular punctures (3–6 s) performed during the first steps of host plant colonisation process (Powell et al., 2006). Therefore, successful vectoring depends firstly on the acquisition access period (AAP) that corresponds to the time elapsed from the aphid contact with the infected plant to the first intracellular puncture.
64
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
Basically, two methods have been used to assess vector efficiency. First, aphids are placed in a closed arena containing both infected and healthy plants (Kanavaki et al., 2006; Katis et al., 2006). However, such a set-up does not allow us to distinguish the contribution of aphid behaviour in the transmission process (vector activity) from their intrinsic ability to transmit viruses (vector efficiency). The second experimental approach aims to control the transmission process by placing the aphid vector on an infected plant for an AAP, and then transferring it to a healthy plant for an inoculation access period (IAP). While the latter method minimizes vector activity contribution as the vectors are artificially placed on the source and then on the target plant, they are usually performed with short AAPs that can induce bias in the vector efficiency assessment. Indeed, it has been demonstrated that AAP varies depending on the aphid species and is modulated by the healthy or infected status of the plant (Boquel et al., 2011). The main objective of this paper was to develop, on the aphid vector – PVY – potato pathosystem, an alternative method to optimize REF assessment by minimizing the part of the source and target plants and the part of vector. In vitro micropropagated potato plantlets were used to get phenotypically and genetically homogeneous plant material (Acquaah, 2007; Hu and Wang, 1983) and minimize potato plant defence responses (Péros et al., 1994; Petroviˇc et al., 1997), and clones of seven aphid species (M. euphorbiae, M. persicae, Rhopalosiphum padi, Sitobion avenae, Brevicoryne brassicae, Aphis fabae and Acyrthosiphon pisum) were used to minimize intraspecific variability. To ensure the vector a sufficient contact time with the plant to perform intracellular puncture and acquire virus particles, electrical penetration graph (EPG) technique was used to define a specific AAP for each aphid species. Finally, this method was used to test the hypothesis suggested by DiFonzo et al. (1997) who postulated that a weak vector efficiency could be compensated by high vector densities in fields.
(INRA-INSA-CIRAD, Montpellier). Each aphid clone, started from a virus free single apterous parthenogenetic female, was reared on a healthy host plant enclosed in a ventilated Plexiglas® cage (360 mm × 240 mm × 110 mm) in a growth chamber (20 ± 1 ◦ C, 60 ± 5% relative humidity, 16:8 h day:night cycle). M. persicae and M. euphorbiae were reared on potato (S. tuberosum L.), A. fabae and A. pisum on broad bean (Vicia faba L.), S. avenae and R. padi on wheat (Triticum aestivum L.) and B. brassicae on broccoli (Brassica oleracea L.). All experiments were performed at 20 ± 1 ◦ C with alates synchronized in host plant seeking phase according to the set-up described by Brunissen et al. (2009).
2.3. Evaluation of a species-specific acquisition access period (AAP) The DC-electrical penetration graph (EPG) technique (Tjallingii, 1978, 1988) was used to determine the necessary time for each aphid species to perform the first potential drop (intracellular puncture) on a PVY-infected in vitro potato plantlet. To insert one aphid and one plant in an electrical circuit, a thin gold wire (20 m diameter, 2 cm long) was stuck with conductive water based silver glue (EPG-systems, Wageningen, The Netherlands) on the aphid’s dorsum and a second electrode was inserted into the agar-based MS medium. The aphid was then connected to the DC-EPG amplifier and placed carefully on a PVY-infected in vitro plantlet into a Faraday cage at an ambient temperature of 20 ± 1 ◦ C. For each aphid species 20 replicates were done. The recordings were performed during daytime for 8 continuous hours. Acquisition and analysis of the EPG waveforms were done with PROBE 3.5 software (EPG-Systems, Wageningen, The Netherlands) and AAP, defined as the mean time from start of the monitoring (when the aphid was deposited on the leaflet) to the first potential drop added to its standard error, was calculated using the EPG-Calc 4.9 software (Giordanengo, 2009).
2. Materials and methods 2.1. Virus isolate and in vitro plantlets The PVYNTN isolate was obtained from potato tubers collected in a potato field (Cambrai, France). Healthy and PVYNTN -infected in vitro potato (cv. Bintje) lines were obtained from germ fragments (2–3 cm), respectively, collected from a healthy and an infected tuber, washed during 20 min with a 7% calcium hypochlorite solution, dried with blotting paper and deposited in a Murashige and Skoog (1962) (MS basal medium with sucrose and agar, 42.4 g L−1 ) medium for development. For micropropagation, explants were isolated on MS medium in a small glass vial (5 mL) placed in a sterile culture glass tube (25 mm × 150 mm) in a growth chamber at 20 ± 1 ◦ C, 60 ± 5% relative humidity and 16:8 h day:night cycle. Each small glass vial containing one 15 day-old healthy or PVYinfected in vitro plantlet was withdrawn from the glass tube for experiments. Immunocapture RT-PCR technique was used to determine the healthy or infected status of the plantlet according to Glais et al. (1998). 2.2. Aphids Aphid species used for virus transmission bioassays were chosen according to their abundance in potato fields in northern France or their ability to transmit PVY (Harrington and Gibson, 1989; Sigvald, 1984; van Harten, 1983; van Hoof, 1980). Aphids were obtained from different French localities or laboratories: M. persicae (potato field, Loos-en-Gohelle), M. euphorbiae (INRAINSA, Villeurbanne), A. fabae (eggplant greenhouse, Amiens), S. avenae and R. padi (INRA, Le Rheu), A. pisum and B. brassicae
2.4. Transmission experiments and relative efficiency factors (REFs) evaluation For each aphid species, 5 synchronized alates were deposited on a PVY-infected in vitro potato plantlet placed in a Plexiglas® box (120 mm × 90 mm × 50 mm) during a period equal to their specific AAP. Then, aphids were deposited individually on a healthy in vitro plantlet grown in the culture glass tube for a 24 h IAP. Finally, after IAP, aphids and their possible progeny were removed and in vitro plantlets were placed in a growth chamber for a 15-day-incubation period before checking for PVY infection by immunocapture RTPCR. For each aphid species, 30 individuals were tested. The REF of each aphid species was calculated by dividing the number of infected plants obtained with the considered species by the number of infected plants obtained with M. persicae used as referent species.
2.5. Number vs efficiency DiFonzo et al. (1997) hypothesized that the high numbers of non-colonizing aphids could compensate their weak virus transmission efficiency. In accordance to the obtained results in transmission experiments, this hypothesis was tested with the two aphid species that ranked, respectively, second and third in the calculated REF. The number of potentially viruliferous aphids deposited simultaneously per target plantlet for IAP was calculated according to the equation: number aphid to use = REF M. persicae/REF tested species. Then, transmission efficiency experiments were realised according to the method described above.
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
65
Table 1 Species-specific acquisition access period (AAP) determined for seven aphid species with electrical penetration graph technique during an-8-h recording on PVY infected in vitro S. tuberosum cv. Bintje plantlets.
Fpd ± SE (min) AAP (min)
M. euphorbiae
M. persicae
A. fabae
R. padi
A. pisum
S. avenae
B. brassicae
12 ± 3 ab 15
9±2 a 11
72 ± 18 d 90
25 ± 6 cd 31
20 ± 10 abc 30
28 ± 7 bcd 35
37 ± 11 cd 48
Different letters indicate significant difference at p < 0.05 according to a Mann–Whitney test after a Kruskal–Walllis test. Fpd, time from start of the recording to the first intracellular puncture (i.e. potential drop); SE, standard error.
2.6. Data analysis Statistical analyses were performed using STATISTICA 8.0 software (StatSoft, Tulsa, OK, USA). Transmission rates were calculated as percentages of infected plants and were compared with the different aphid species using a Chi-square test. Because EPG data were not normally distributed, pairwise comparisons were done between the different aphid species with a Mann–Whitney U-test after a global analyse with the Kruskal–Wallis (H) test. Significant differences were determined at p < 0.05. 3. Results 3.1. Evaluation of species-specific acquisition access period (AAP) For each aphid species, different AAP were obtained (Table 1). M. persicae showed the shortest AAP (11 min) followed by M. euphorbiae (15 min). AAP of non-colonizing aphid species varied from 30 min for A. pisum to 48 min for B. brassicae except for A. fabae which took 90 min to realise the first potential drop. 3.2. Transmission experiments and relative efficiency factors (REFs) evaluation Most aphid species tested transmitted PVYNTN isolate with different transmission rates. M. persicae exhibited the greatest rate of transmission with 83.3% and its REF was set to 1 (Table 2). The other potato colonizing aphid M. euphorbiae exhibited a significant lower transmission rate (26.7%, REF = 0.32, 2 = 17.24, p < 0.01) than that of M. persicae. Among non-colonizing aphids species, S. avenae and A. pisum showed intermediate transmission rates (16.7%, REF = 0.20 and 6.7%, REF = 0.08, respectively), which only significantly differed from that of M. persicae (2 = 24.07, p < 0.01; 2 = 32.59, p < 0.01, respectively). The three remaining species, B. brassicae (3.3%, REF = 0.04), A. fabae (0%, REF = 0.00) and R. padi (0%, REF = 0.00), exhibited significant lower transmission rates compared to that of both potato colonizing aphids M. persicae (2 = 35.90, p < 0.01; 2 = 39.50, p < 0.01; 2 = 39.50, p < 0.01, respectively) and M. euphorbiae (2 = 4.71, p < 0.05; 2 = 7.07, p < 0.01; 2 = 7.07, p < 0.01, respectively).
Table 2 PVY transmission efficiency and relative efficiency factors (REFs) of seven aphid species. Aphid species
Number of aphid per plant
Infected plantlets (%)
REF
M. persicae M. euphorbiae A. pisum A. fabae B. brassicae R. padi S. avenae
1 1 1 1 1 1 1
83.3 a 26.7 b 6.7 bc 0c 3.3 c 0c 16.7 bc
1.00 0.32 0.08 0.00 0.04 0.00 0.20
Different letters indicate significant difference between species at p < 0.05 according to a Chi-square test. REFs were calculated according to the equations describe in Materials and methods section (2.4).
Table 3 PVY transmission efficiency and relative efficiency factors (REFs) of 3 aphid species according to the number of aphids used to infect target in vitro S. tuberosum cv. Bintje plantlets. Aphid species
Number of aphid per plant
Infected plantlets (%)
REF
M. persicae M. euphorbiae S. avenae
1 3 5
83.3 a 3.3 b 23.3 b
1.00 0.04 0.28
Different letters indicate significant difference between species at p < 0.05 according to a Chi-square test. Number of aphid per plant and REFs were calculated according to the equations describe in Materials and methods section (2.5 and 2.4, respectively).
3.3. Number vs efficiency As M. euphorbiae and S. avenae ranked respectively second and third in REF, the number of potentially viruliferous aphids to use for IAP was calculated for both species. It was nearly equal to 3 for M. euphorbiae and 5 for S. avenae. Results of transmission efficiencies showed that for neither of the two species (M. euphorbiae: 3.3%, 2 = 35.90, p < 0.01 and S. avenae: 23.3%, 2 = 19.35, p < 0.01) did a higher number of vectors compensate their weak transmission rate compared to M. persicae (Table 3). Surprisingly, increasing the number of viruliferous aphids on a target plantlet during the IAP led to reduced transmission efficiency for M. euphorbiae (3.3% vs 26.7%, 2 = 4.71, p < 0.05) and an unchanged transmission efficiency for S. avenae (23.3% vs 16.7%, 2 = 0.10, p = 0.75). 4. Discussion With the objective to evaluate the vector efficiency of aphid species, REF values can be estimated reliably by assessing transmission rates of individual aphids using species-specific AAPs and in vitro plantlets as inoculum source and target plants. The transmission rate obtained for M. persicae (83.3%) was higher than the one reported in the literature irrespective of the M. persicae clone used (Halbert et al., 2003; Sigvald, 1984; Verbeek et al., 2010). It was also higher than the one obtained previously for the same M. persicae clone (45%) on plants grown from tubers from the same S. tuberosum cultivar Bintje (data not shown). Such discrepancy observed in transmission efficiency could be attributed to the plant material used (in vitro vs tuber-grown potato plants) rather than to the aphid clone used. Compared to tuber-grown plants, in vitro plantlets may be unable to develop efficient defence responses towards pathogens (Péros et al., 1994; Petroviˇc et al., 1997) thus leading to successful virus infection. Consequently, one could expect higher virus concentration in in vitro material as reported by Petroviˇc et al. (1997) and a more homogeneous distribution of viral particles within plantlet tissues. Moreover, as confirmed by the semiquantitative RT-PCR performed on in vitro potato plantlets used as source for AAP in the transmission experiments, virus concentrations were constant and no significant difference was measured between plantlet series (data not shown). Finally, the RT-PCR technique used in this study allows a more accurate assessment of the infected status of the plant as the detection threshold of viral particles is lower than the commonly used ELISA technique (Moreno et al., 2007; Wang and Ghabrial, 2002).
66
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67
The EPG monitoring revealed that the time elapsed from the aphid contact with the infected plant until the first intracellular puncture (AAP), necessary for virus acquisition (Powell et al., 1995), differed between aphid species. M. persicae and M. euphorbiae took less than 15 min to achieve the first intracellular puncture while non-colonizing aphid species needed more than 30 min to exhibit such behaviour. These different AAPs between colonizing and non-colonizing aphids could result from their host plant range. M. persicae and M. euphorbiae are polyphagous aphids and S. tuberosum is one of their favoured secondary hosts (Blackman and Eastop, 2000). By contrast, as potato is not a host plant for the non-colonizing aphid species (Blackman and Eastop, 2000), delayed times in their first intracellular puncture could be associated to a lower plant acceptance. The variations in AAPs observed could also be due, at least partly, to the infected status of the plant. It is assumed that plant infection by phytoviruses can affect behaviour and physiology of vectoring aphids (Fereres and Moreno, 2009). On PVY-infected potato plantlets, M. persicae exhibited increased phloem ingestion and reduced non-probing duration whereas M. euphorbiae showed delayed stylet insertion, reduced activity in the phloem vessels and an enhanced nonprobing duration (Boquel et al., 2011). These results suggested that these behavioural variations might be a consequence of a physical modification or the induction of chemical compounds (e.g. antifeedant/phagostimulant) in vascular and mesophyll tissues. Such results underline that it is crucial to measure species-specific AAP in order to reliably evaluate vector efficiency. In most of the published data, a single AAP of generally 5 min after proboscis contact was used whatever the aphid species (Fernández-Calvino et al., 2006; Katis et al., 2006; Verbeek et al., 2010) leading to underestimated REFs. Indeed, proboscis contact with the plant did not revealed an efficient acquisition, as intracellular puncture was not highlighted. In this work, a method using the EPG tool that allows estimation of a more accurate AAP was proposed. These experiments confirmed M. persicae as the most efficient vector of PVY. M. euphorbiae appeared as an efficient vector with 26.7% transmission rate whereas it was reported as a weak or a non vector of PVY (Harrington and Gibson, 1989; Singh and Boiteau, 1986; Verbeek et al., 2010). Whatever the clone tested, no or very low transmission rates were observed with S. avenae (de Bokx and Piron, 1990; Piron, 1986; van Hoof, 1980; Verbeek et al., 2010). Surprisingly, a high PVY REF was obtained for S. avenae clone, suggesting its significant involvement in the spread of PVY. In accordance with the literature, R. padi, A. fabae, B. brassicae and A. pisum showed very low or even undetectable transmission rates (de Bokx and Piron, 1990; Harrington and Gibson, 1989; Heimbach et al., 1998; Piron, 1986; Verbeek et al., 2010). DiFonzo et al. (1997) suggested that aphids showing weak vector efficiencies might play an important role in the epidemics of non-persistent viruses provided that their field population densities are high. In these experimental conditions, increasing the number of viruliferous aphids deposited on the target plant did not enhance the transmission efficiencies and then did not corroborate this hypothesis. The REF of S. avenae was unchanged by increasing the number of viruliferous aphids set on a plant. Surprisingly, increasing the number of M. euphorbiae set on a healthy plant during the IAP decreased its vector efficiency. M. euphorbiae is a fairly mobile species (Boiteau, 1997) and did not settle easily on the small in vitro plantlets (pers. obs.) which could reduce virus transmission. Moreover, a high aphid number could enhance the plantlet defence responses as reported on tuber-grown potato plants (Ameline et al., 2007; Dugravot et al., 2007). This could explain, at least partly, the low efficiency rates reported in the previous works where up to 5 aphids were used per target plant (Kalleshwaraswamy and Krishna-Kumar, 2007; Katis et al., 2006; van Hoof, 1980). In a methodological context, using only one aphid
as a vector on the target in vitro plantlet seems to be sufficient to estimate the REF and would avoid bias consequent to intraspecific interactions. However, because of the limitations imposed by the in vitro model preventing the use of numerous aphids, the DiFonzo et al. (1997) hypothesis was not tested with either aphids exhibiting low vector efficiencies or with higher numbers of M. euphorbiae or S. avenae. Brought together with the high viral infection rates even in the absence of colonizing aphids which exhibit higher transmission efficiencies, these results cannot lead to preclude that successful spread of PVY in the fields could result from the massive densities of non colonizing aphids. Therefore, it would be worth including other parameters such as interplant movements to validate this hypothesis since non-colonizing aphid species are more restless than colonizing ones (Nemecek et al., 1993). To conclude, a reliable method to evaluate PVY transmission efficiency is proposed. Species-specific AAPs evaluation insures optimal virus acquisition and using in vitro plants allows saving time and space regarding their rapid growth and small size. With the aim to develop a reliable method to assess REFs, aphid material was standardized by using only one clone. As variations in transmission rates can occur between different clones (Woodford, 1992), epidemic studies would integrate such intraspecific variations and compare the vector efficiency of several clones to improve REF assessment of the considered species.
Acknowledgements This work was funded by the Conseil Régional de Picardie and the Comité Nord Plants de Pommes de Terre. We thank D. Tagu (INRA, Rennes, France) for providing the S. avenae and R. padi clones, M. Uzest (INRA-CIRAD, Montpellier, France) for A. pisum and B. brassicae clones, Y. Rahbé (INRA-INSA, Villeurbanne) for M. euphorbiae clone and F. Lemoine and X. Riquiez from the Comité Nord Plants de Pommes de Terre for providing healthy in vitro plant lineage and infected tuber. The authors also thank Aude Couty, Andrew Roots and Mamed Ferkioui for their advice in the usage of the English language.
References Acquaah, G., 2007. Principles of Plant Genetics and Breeding. Blackwell Publishing, Oxford. Ameline, A., Couty, A., Dugravot, S., Campan, E., Dubois, F., Giordanengo, P., 2007. Short-term alteration of Macrosiphum euphorbiae host plant selection behaviour after biotic and abiotic damages inflicted to potato plants. Entomol. Exp. Appl. 123, 129–137. Blackman, R.L., Eastop, V.F., 2000. Aphids on the World’s Crop: An Identification and Information Guide, Second ed. John Wiley & Soons LTD, New York. Boiteau, G., 1997. Comparative propensity for dispersal of apterous and alate morphs of three potato-colonizing aphid species. Can. J. Zool. 75, 1396–1403. Boiteau, G., Singh, M., Singh, R.P., Tai, G., Turner, T., 1998. Rate of spread of PVYN by alate Myzus persicae (Sulzer) from infected to healthy plants under laboratory conditions. Potato Res. 41, 335–344. Boquel, S., Giordanengo, P., Ameline, A., 2011. Divergent effects of PVY-infected potato plant on aphid. Eur. J. Plant Pathol. 129, 507–510. Brunissen, L., Cherqui, A., Pelletier, Y., Vincent, C., Giordanengo, P., 2009. Host–plant mediated interactions between two aphid species. Entomol. Exp. Appl. 132, 30–38. de Bokx, J.A., Piron, P.G.M., 1990. Relative efficiency of a number of aphid species in the transmission of Potato virus YN in the Netherlands. Neth. J. Plant Pathol. 96, 237–246. DiFonzo, C.D., Ragsdale, D.W., Radcliffe, E.B., Gudmestad, N.C., Secor, G.A., 1997. Seasonal abundance of aphid vectors of Potato virus Y in the Red River Valley of Minnesota and North Dakota. J. Econ. Entomol. 90, 824–831. Dugravot, S., Brunissen, L., Letocart, E., Tjallingii, W.F., Vincent, C., Giordanengo, P., Cherqui, A., 2007. Local and systemic responses induced by aphids in Solanum tuberosum plants. Entomol. Exp. Appl. 123, 271–277. Fereres, A., Moreno, A., 2009. Behavioural aspects influencing plant virus transmission by homopteran insects. Virus Res. 141, 158–168. Fereres, A., Perez, P., Gemeno, C., Ponz, F., 1993. Transmission of Spanish pepper- and potato-PVY isolates by aphid (Homoptera: Aphididae) vector: epidemiological implications. Environ. Entomol. 22, 1260–1265.
S. Boquel et al. / Journal of Virological Methods 178 (2011) 63–67 Fernández-Calvino, L., López-Abella, D., López-Moya, J.J., Fereres, A., 2006. Comparison of Potato virus Y and Plum pox virus transmission by two aphid species in relation to their probing behavior. Phytoparasitica 34, 315–324. Giordanengo, P., 2009. EPG-Calc: A PHP Program to Evaluate Epg Parameters. Université de Picardie Jules Verne, Available online: http://www.upicardie.fr/PCP/UTIL/epg.php. Glais, L., Tribodet, M., Gauthier, J.P., Astier-Manifacier, S., Robaglia, C., Kerlan, C., 1998. RFLP mapping of the whole genome of ten viral isolates representative of different biological groups of Potato virus Y. Arch. Virol. 143, 2077–2091. Halbert, S.E., Corsini, D.L., Wiebe, M.A., 2003. Potato virus Y transmission efficiency for some common aphids in Idaho. Am. J. Potato Res. 80, 87–91. Hamm, P.B., Hane, D.C., Pavek, M.J., Leroux, L.D., Gieck, S.L., David, N.L., 2010. Potato cultivars differ in current season Potato Virus Y (PVY) infection. Am. J. Potato Res. 87, 19–26. Harrington, R., Gibson, R.W., 1989. Transmission of Potato virus Y by aphids trapped in potato crop in southern England. Potato Res. 32, 167–174. Heimbach, U., Thieme, T., Weidemann, H.L., Thieme, R., 1998. Transmission of Potato virus Y by aphid species which do not colonise potatoes. In: Nieto-Nafria, J.M., Dixon, A.F.G. (Eds.), Aphids in natural and managed ecosystems. Universitad de León, León, pp. 555–559. Hu, C.Y., Wang, P.J., 1983. Meristem shoot tip and bud cultures. In: Evans, D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. 1. MacMillan Publishing Company, New York, pp. 177–227. Kalleshwaraswamy, C.M., Krishna-Kumar, N.K., 2007. Transmission efficiency of Papaya ringspot virus by three aphid species. Phytopathology 98, 541–546. Kanavaki, O.M., Margaritopoulos, J.T., Katis, N.I., 2006. Transmission of Potato virus Y in tobacco plants by Myzus persicae nicotianae and M. persicae s.str. Plant Dis. 90, 777–782. Katis, N.I., Tsitsipis, J.A., Lykouressis, D.P., Papapanayotou, A., Margaritopoulos, J.T., Kokinis, G.M., Perdikis, D.C., Manoussopoulos, I.N., 2006. Transmission of Zucchini yellow mosaic virus by colonizing and non-colonizing aphids in Greece and new aphid species vectors of the virus. J. Phytopathol. 154, 293–302. Lupoli, R., Labonne, G., Yvon, M., 1992. Variability in the transmission efficiency of potyviruses by different clones of Aphis gossypii. Entomol. Exp. Appl. 65, 291–300. Moreno, A., Bertolini, E., Olmos, A., Cambra, M., Fereres, A., 2007. Estimation of vector propensity for Lettuce mosaic virus based on viral detection in single aphids. Span. J. Agric. Res. 5, 376–384. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.
67
Nemecek, T., Fischlin, A., Derron, J., Roth, O., 1993. Quantifying behaviour sequences of winged aphids on potato plants for virus epidemic models. Bull. Entomol. Res., 1–16. Péros, J.P., Bonnel, E., Roques, D., Paulet, F., 1994. Effect of in vitro culture on rust resistance and yield in sugarcane. Field Crops Res. 37, 113–119. Petroviˇc, N., Miersch, O., Ravnikar, M., Kovaˇc, M., 1997. Potato virus YNTN alters the distribution and concentration of endogenous jasmonic acid in potato plants grown in vitro. Physiol. Mol. Plant Pathol. 50, 237–244. Piron, P.G.M., 1986. New aphid vectors of Potato virus YN . Neth. J. Plant Pathol. 92, 223–229. Pirone, T.P., Megahed, E.S., 1966. Aphid transmissibility of some purified viruses and viral RNAs. Virology 30, 631–637. Powell, G., Pirone, T.P., Hardie, J., 1995. Aphid stylet activities during potyvirus acquisition from plants and an in vitro system that correlate with subsequent transmission. Eur. J. Plant Pathol. 101, 411–420. Powell, G., Tosh, C.R., Hardie, J., 2006. Host plant selection by aphids: behavioral, evolutionary, and applied perspectives. Annu. Rev. Entomol. 51, 309–330. Radcliffe, E.B., Ragsdale, D.W., 2002. Aphid-transmitted potato viruses: the importance of understanding vector biology. Am. J. Potato Res. 79, 353–386. Sigvald, R., 1984. The relative efficiency of some aphid species as vectors of Potato virus YO (PVYO ). Potato Res. 27, 285–290. Sigvald, R., 1992. Progress in aphid forecasting systems. Neth. J. Plant Pathol. 98, 55–62. Singh, R.P., Boiteau, G., 1986. Reevaluation of the potato aphid, Macrosiphum euphorbiae (Thomas), as vector of Potato virus Y. Am. J. Potato Res. 63, 335–340. Tjallingii, W.F., 1978. Electronic recording of penetration behaviour by aphids. Entomol. Exp. Appl. 24, 521–530. Tjallingii, W.F., 1988. Electrical recording of stylet penetration activities. In: Minks, A.K., Harrewijn, P. (Eds.), Aphids: Their Biology, Natural Enemies and Control, vol. 2B. Elsevier, Amsterdam, pp. 95–108. van Harten, A., 1983. The relation between aphid flights and the spread of Potato virus YN (PVYN ) in the Netherlands. Potato Res. 26, 1–15. van Hoof, H.A., 1980. Aphid vectors of Potato virus YN . Neth. J. Plant Pathol. 86, 159–162. Verbeek, M., Piron, P.G.M., Dullemans, A.M., Cuperus, C., van der Vlugt, R.A.A., 2010. Determination of aphid transmission efficiencies for N. NTN and Wilga strains of Potato virus Y. Ann. Appl. Biol. 156, 39–49. Wang, R.Y., Ghabrial, S.A., 2002. Effect of aphid behavior on efficiency of transmission of Soybean mosaic virus by the soybean-colonizing aphid, Aphis glycines. Plant Dis. 86, 1260–1264. Woodford, J.A.T., 1992. Virus transmission by aphids in potato crops. Neth. J. Plant Pathol. 2, 47–54.