Identification and biological characterization of new viral pathogens affecting fruit trees

Fruit trees are high value crops which can be infected by viruses/viroids that tend to accumulate due to the vegetative propagation of the plants and the grafting. Some of the viruses cause similar symptoms, and laboratory protocols need to be applied for a specific identification, while other diseases still have an unknown etiology. The identification of the pathogenic viral species and their biological characterization are key information to control potential epidemics of viral diseases. With the advent of high throughput sequencing (HTS) technologies, the pace of discovery of new viral species in symptomatic plants has accelerated. More than 100 virus/viroid species have been reported from fruit trees the last decade. These viruses have been identified from samples presenting symptoms of unknown etiology but also from other symptomatic samples tested negative for other known plant viruses. HTS technologies will allow the reconstruction of partial or complete virus genome without any information on the biology of the virus. Biological characterization of a virus is indeed a complex and long process. These new virus discoveries, sometimes corresponding to the identification of a new viral genome only, have widened the gaps in our understanding of virus biology. The two main objective(s) of this thesis are (i) to critically review the newly discovered fruit trees viruses for answering the question: how biological characterization of a new virus could follow the current pace of virus discovery and to suggest prioritization of complementary experiments to be carried out once a new plant virus is discovered in the near future, and (ii) to report the identification and genome characterization of a newly reported virus, apple necrotic mosaic virus (ApNMV), from a new natural host: hawthorn(Crataegus spp) on a tree presenting mosaic symptom.
(1) A retrospective analysis for new fruit tree viruses
The review analyzed 78 publications of new viruses and viroids discovered from 32 fruit tree species since 2011. The biological information useful for a pest risk assessment and published together with the discovery of a new fruit tree virus or viroid has been analyzed taking into account a biological characterization framework proposed for new plant viruses. In addition, the 933 publications citing at least one of these original publications were reviewed, focusing on the biology-related information provided.
In the original publications, biological characterization experiments only had been selectively carried out, the scientific information provided was the development of a detection test (94%), the whole genome sequence including UTRs (92%), local and large-scale epidemiological surveys (68%), infectivity and indicators experiments (50%), association study with symptoms (25%), host range (23%) and transmission mode or vector identification (8%). Twenty percent of the new virus species have been published without any information on their biological characterization but only genome information.
The publication of a new virus is cited 2.8 per year on average. Only 18% of the citations brought information on the biology or geographical repartition of the new viruses. These citing publications improved only slightly the new virus characterization, and mainly on enlarging the geographical distribution (11%) or host range (5%).
Based the gathering of scientific information on the virus biology in original and citing publications, the fulfilment of recently proposed framework has been evaluated. Long term biological characterization experiments have been carried out in global epidemiology (61%), transmission and vectors (44%), symptoms and host range (55%). In conclusion, the biology of most of these newly discovered viruses and the associated risks for plant health remain largely unknown.
Overall, the biological characterization of a newly discovered virus on fruit tree species is rarely pursued after the first discovery, which reinforce the need to bring as much information as possible when publishing a new virus, even though the biological characterization experiments can be time-consuming and could delay the publication of the results. Minimal recommendations for publishing a new fruit tree virus and prioritization of complementary experiments to be carried out were suggested for any new fruit tree discovery in the future.
(2) The case of a new viral pathogen: apple necrotic mosaic virus identified from apple trees, possibly associated with hawthorn mosaic disease
Apple mosaic disease is widespread in the major apple-producing areas in China, and frequently associated with the presence of the newly identified apple necrotic mosaic virus (ApNMV), belonging to subgroup 3 of Ilarvirus genus in the family of Bromoviridae. Mosaic symptoms were also observed in hawthorn tree. HTS technologies revealed the hawthorn tree with mosaic symptom was infected by ApNMV, which was confirmed by RT-PCR. The complete nucleotide sequences of RNA1 (3,378 nt), RNA2 (2,778 nt) and RNA3 (1,917 nt) of ApNMV from the hawthorn were obtained, sharing 93.8 - 96.8%, 89.7 - 96.1% and 89.8 - 94.6% nucleotide identities with those from apples and crab apples, respectively. Two hypervariable regions were found which showed 59.2 - 85.7% and 64.0 - 89.3% sequence identities at position 142 - 198 aa and at position 780 - 864 aa in the POL protein, respectively, between the hawthorn isolate and other isolates (apple, crabapple). Grafting test demonstrated ApNMV was easily transmissible from hawthorns to apple trees with severe chlorosis, yellowing, mosaic, curling and necrosis. In addition, a total of 11,685 hawthorn trees were surveyed for the incidence of mosaic disease from five provinces in China, only six were showing typical mosaic symptoms. A total of 145 individual trees (6 symptomatic, 68 asymptomatic and 71 with other symptoms) were tested for the presence/absence of ApNMV by RT-PCR. Among them, 6 symptomatic, 4 asymptomatic and 10 other symptomatic trees tested positive for ApNMV. Taken together, these results demonstrated that the hawthorn tree was identified as a new natural host for ApNMV with a relatively low frequency (13.8%, 20 out of 145) in the main producing areas, and it was likely to be one of the causal pathogens of hawthorn mosaic disease.