Virus Q&A

Questions and Answers
Regarding the Discovery of CaMMV and CYVBV in ICQC,R February 2022

Background

One of the greatest challenges facing any plant quarantine centre is the detection of viruses since some virus species can remain latent for long periods or cause very mild or no symptoms in their hosts. The International Cocoa Quarantine Centre at the University of Reading (ICQC,R) uses an internationally accepted virus-indexing procedure developed to detect Cocoa Swollen Shoot Virus Disease (CSSVD), a disease that causes severe crop losses and tree death in many cocoa producing areas of West Africa. Clearly the spread of CSSVD to additional cocoa growing areas would have very serious consequences for the whole of the global cocoa industry, thus underlying the importance of effective quarantine when moving live cocoa plant material into any cocoa producing country.

Although some mild cocoa viruses, that are distinct from CSSVD, have been reported from areas outside of West Africa, the extent of their distribution has remained largely unknown. It has been difficult to confirm infections since these mild viruses cause few or no symptoms, or produce symptoms which are similar to those caused by some nutrient deficiencies. However, recent developments in molecular methods have allowed the recent detection of two mild strain viruses, Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein Banding Virus CYVBV and have led to the discovery of these viruses in various locations (Puig et al. 2020; Puig 2021; Ramos-Sobrinho et al. 2021) including in the International Cocoa Quarantine Centre, Reading (ICQC,R) (Ullah et al. 2021).

The following Questions and Answers seek to provide clarity on the broader implications of this recent finding.

Q&A

Cacao Mild Mosaic Virus (CaMMV) and Cacao Yellow Vein Banding Virus (CYVBV) were named following molecular characterization of two badnaviruses in samples of cocoa trees with some visible symptoms seen in the International Cocoa Genebank, Trinidad (ICG,T) (Sreenivasan 2009; Chingandu et al. 2017). These viruses correspond to two distinctive virus like symptoms detected in Trinidad in the 1940s and then putatively designated as being caused by Cocoa Trinidad Virus (CTV) Strain A and Strain B, based on their symptomology; CTV-Strain A (now CaMMV) causing red foliar mottling and CTV-Strain B (now CYVBV) causing yellow vein-banding on cocoa leaves (Posnette, 1944; Baker and Dale 1947a; Baker and Dale 1947b; Chingandu et al. 2017). CaMMV can also cause red/purple mottling on pods. Three isolates ('strains') of CaMMV have recently been shown to exist (Ramos-Sobrinho et al. 2021).
CaMMV and CYVBV are mild strain viruses that are distinct from any of the virus species known to be associated with Cocoa Swollen Shoot Virus Diseases (CSSVD). Although CaMMV and CYVBV can cause leaf symptoms in some cocoa genotypes they are not associated with stem swellings and all the evidence available to date is that they do not have the severe impact on the tree or on the crop that is seen with CSSVD (see Muller 2017, for a description of CSSVD).
All the clones that have tested positive at ICQC,R using the molecular tests were asymptomatic (Ullah et al. 2021). Elsewhere, some mild symptoms have been observed. Historical reports of the virus outbreak in Trinidad from the 1940s suggest an impact of these viruses on vigour and yield for some of the Trinitario varieties widely grown there at the time (Baker and Dale 1947a). However, further detailed research would be needed to determine whether CaMMV and CYVBV have the same impact, whether some varieties are more susceptible to one or both viruses than others and how environmental factors (such as temperature, drought, soil nutrient availability) might influence expression of viral symptoms.
Four clones tested positive for CaMMV (GEBP 584/A-F, PNG 138, SCA 12, WA 40) and eleven for CYVBV (GU 144/C, GU219/F, GU 265/P, GU 341/H, IMC 85, JA 10/12, ICS 60, ICS 95, NAP 25, PA 156, TRD 45) from the 350 genotypes tested (and which had previously passed quarantine procedures) at ICQC,R.
CaMMV and CYVBV are freely replicating viruses that can be transmitted between plants by means of a vector (mealybugs are currently the only known vector). We were involved in the research that led to the recent report (Muller et al. 2021) that describes the discovery of integrated viral sequences in cocoa that seemingly do not spread between plants or have any impact on the plant.
The recent reports of positive molecular tests for CaMMV and CYVBV in cocoa samples from South America, Caribbean and Southeast Asia (Probowati et al., 2019) suggest that both viruses could be quite widely distributed though information is still lacking on their exact distribution. Since the plants infected with these viruses can remain asymptomatic, it seems highly likely that historically the viruses have evaded detection in material passing through various quarantine routes, though it is not possible to determine the route, or routes, by which the viruses have spread. Recent research suggests that although their most recent source was from Trinidad, it is possible that these viruses may have their origin in South America and have then become widespread globally over the last century (Ullah et al. 2021). However, more research is needed on how widespread these viruses are and whether they are detected in cocoa’s centre of origin - in the rainforests of the Amazon.
Although the virus-indexing procedures used by ICQC, R are designed to detect CSSV, they have also been successful in detecting non-CSSV viruses. In 2004, a virus was detected in the variety ('clone') ICS 76 whilst it was undergoing quarantine (Sreenivasan, 2009), through the detection of visual symptoms on 'test plants' (grafted plants of a variety known to be susceptible to viruses) as part of the quarantine process. The clone had been received from ICG,T and this led to renewed interest in the occurrence of cocoa viruses in Trinidad and the subsequent characterisation of CaMMV and CYVBV by Chingandu et al. (2017).
The clones in which the viruses were recently detected did not cause symptoms either in the mother plants or the grafted test plants. In cases of asymptomatic viral infection, the ability to detect the virus is limited by knowledge and technology available at any given time. We have been able to detect these viruses recently in a broader range of materials due to advances in the technology that we have now routinely adopted at ICQC,R (see below).
  1. Two types of laboratory tests ('PCR' and the more recently developed 'LAMP' test) will be conducted on all plants in ICQC,R at regular intervals.
  2. Additional test plants are being used in screening for viruses using a variety known to be susceptible to CaMMV and CYVBV.
  3. Mealybugs are the known vector of cocoa viruses. No mealybugs are present in the ICQC,R facilities and very strict measures are in place to prevent any such vectors entering the facilities.
  4. Whilst the virus can be spread through grafting, it is not known whether cocoa viruses can be spread via pruning and grafting tools. Nevertheless, to negate this possibility, a strict regime of tool cleaning has been adopted in all processes in ICQC,R and is in in daily use within the facilities.
Work is underway to better understand how the viruses are distributed within the plant. However, it is already known that they can remain localised and so budwood taken from a plant that has tested positive will not necessarily be infected with the virus. Nevertheless, we would recommend that a molecular test of any material of the 15 aforementioned clones that have been received from ICQC,R is undertaken locally. The method and primers needed are described in Ullah et al. (2021) and Puig (2021). We would be very interested in hearing of the results, which may help inform other recipients of the material. Please advise us of the negative or positive results of any such tests that are completed to the following email address: a.j.daymond@reading.ac.uk
  1. Baker, R.E.D. & Dale, W.T. (1947a) 'Notes on a virus disease of cacao.' Annals of Applied Biology, 34, 60–65.
  2. Baker, R.E.D. & Dale, W.T. (1947b) 'Virus diseases of cacao in Trinidad-II.' Tropical Agriculture, 24, 127-130.
  3. Chingandu, N., Zia-Ur-Rehman, M., Sreenivasan, T.N., Surujdeo-Maharaj, S., Umaharan, P., Gutierrez, O.A., Brown, J.K. (2017) 'Molecular characterization of previously elusive badnaviruses associated with symptomatic cacao in the New World'. Archives of Virology, 162, 1363–1371. DOI: 10.1007/s00705-017-3235-2
  4. Muller, E., Ullah, I., Dunwell, J.M., Daymond, A.J., Richardson, M., Allainguillaume, J., Wetten, A. (2021) 'Identification and distribution of novel badnaviral sequences integrated in the genome of cacao (Theobroma cacao)'. Scientific Reports, 11, 1-13.
  5. Puig, A.S., Ramos-Sobrinho, R., Keith, C., Kitchen, N., Gutierrez, O., Goenaga, R., Brown, J.K. (2020) 'First report of cacao mild mosaic virus associated with symptomatic commercial cacao (Theobroma cacao) trees in Puerto Rico'. Plant Disease, 104, 3089.
  6. Muller, E. (2017) 'Cacao swollen shoot virus (CSSV): History, Biology and Genome.' In: (B.A. Bailey and L.W. Meinhardt Eds.). Cacao Diseases. Springer, Switzerland. pp. 337-360.
  7. Posnette, A. F. (1944) 'Virus diseases of cacao in Trinidad.' Tropical Agriculture, Trinidad and Tobago, 21, 105-6.
  8. Probowati, W., Susamto, S. & Sedyo, H. (2019) 'Molecular characterization of Mosaic Virus from the cocoa trees showing mosaic symptoms in Yogyakarta, Indonesia.' Biodiversitas Journal of Biological Diversity, 20.
  9. Puig, A.S., Ramos-Sobrinho, R., Keith, C., Kitchen, N., Gutierrez, O., Goenaga, R. & Brown, J.K. (2020) 'First report of cacao mild mosaic virus associated with symptomatic commercial cacao (Theobroma cacao) trees in Puerto Rico.' Plant Disease, 104, 3089. DOI: 10.1094/PDIS-04-20-0745-PDN
  10. Puig, A.S. (2021) 'Detection of cacao mild mosaic virus (CaMMV) using nested PCR and evidence of uneven distribution in leaf tissue'. Agronomy, 11(9), 1842. DOI: 10.3390/agronomy11091842
  11. Ramos-Sobrinho, R., Ferro, M.M.M., Nagata, T., Puig, A.S., von Keith, C., Santos Britto, D., Guitierrez, O.A., Marelli, J-P., Brown, J.K. (2021) 'Complete genome sequences of three newly discovered cacao mild mosaic virus isolates from Theobroma cacao L. in Brazil and Puerto Rico and evidence for recombination'. Archives of Virology, 166, 2027-2031.
  12. Sreenivasan, T. N. (2009) 'The enigma of the ICS 76 plants at Reading, U.K.' St Augustine, Trinidad and Tobago. 7-2031.
  13. Ullah, I., Daymond, A.J., Hadley, P., End, M.J., Pathmanathan, U., Dunwell, J.M. (2021) 'Identification of cacao mild mosaic virus (CaMMV) and cacao yellow vein-banding virus (CYVBV) in cocoa (Theobroma cacao) germplasm'. Viruses, 13, 2152. DOI: 10.3390/v13112152

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