Analyses of virus/viroid communities in nectarine trees by next-generation sequencing and insight into viral synergisms implication in host disease symptoms

Abstract

We analyzed virus and viroid communities in five individual trees of two nectarine cultivars with different disease phenotypes using next-generation sequencing technology. Different viral communities were found in different cultivars and individual trees. A total of eight viruses and one viroid in five families were identified in a single tree. To our knowledge, this is the first report showing that the most-frequently identified viral and viroid species co-infect a single individual peach tree, and is also the first report of peach virus D infecting Prunus in China. Combining analyses of genetic variation and sRNA data for co-infecting viruses/viroid in individual trees revealed for the first time that viral synergisms involving a few virus genera in the Betaflexiviridae, Closteroviridae, and Luteoviridae families play a role in determining disease symptoms. Evolutionary analysis of one of the most dominant peach pathogens, peach latent mosaic viroid (PLMVd), shows that the PLMVd sequences recovered from symptomatic and asymptomatic nectarine leaves did not all cluster together, and intra-isolate divergent sequence variants co-infected individual trees. Our study provides insight into the role that mixed viral/viroid communities infecting nectarine play in host symptom development, and will be important in further studies of epidemiological features of host-pathogen interactions.

Figure 1

Disease phenotypes of the five nectarine trees sampled for sRNA sequencing. (A) Cultivar ‘Youtao 1233’ showing typical symptoms on two trees in greenhouse #1, (B) cultivar ‘Zhongyou 4’ showing symptoms on three trees in greenhouse #2.

Figure 2

Confirmation of the identified viruses and viroids by RT-PCR. RT-PCR was used to amplify partial sequences of (A) APV1, (B) APV2, (C) APV3, (D) ACLSV, (E) CGRMV, (F) CNRMV, (G) GRGV, (H) PBNSPaV, (I) PeVD, (J) NSPaV, (K) PaLV, and (L) PLMVd using virus or viroid-specific primer pairs from nectarine tree RNA samples T01, T02, T03, T04, and T05. Amplified DNA fragments were examined by electrophoresis on 1.5% agarose gels and stained with EtBr. M, DNA size 2000 marker; N, negative control.

Figure 3

Identification and quantification of the pathogen-associated sequencing reads for the viruses/viroids infecting tree samples T01 and T02 using sRNA sequencing. (A) The numbers of assembled viral contigs associated with known viruses and viroids in samples T01 and T02 from greenhouse #1. (B) The percentage of virus or viroid-associated reads in each sample was calculated as the number of viral reads divided by the number of clean sRNA sequencing reads (x 100). (C) And by the total number of pathogen reads from all identified viruses and viroids (x 100). The reads from APV1 and APV3 were combined (“APVs”) due to their high degree of sequence similarity. Sequencing reads that aligned to another 11 contigs with identities to marafi-, tymo-, and maculaviruses, corresponding to segments with low sequence similarity that may be a novel unknown virus in the family Tymoviridae, were combined as “other”.

Figure 4

Identification and quantification of the pathogen -associated sequencing reads for the viruses/viroids infecting tree samples T03, T04, and T05 using sRNA sequencing. (A) The numbers of assembled viral contigs associated with known viruses and viroids in samples T03, T04 and T05 from greenhouse #2. The percentage of virus and PLMVd-associated reads (B,C) in each sample was calculated as described in Fig. 3 for samples T03, T04, and T05.

Figure 5

Phylogenetic analysis of PLMVd isolates. The phylogenetic tree was constructed by the maximum-likelihood (ML) method using MEGA6 software. Bootstrap confidence values (1,000 replicates) are given at the branch nodes. Branches corresponding to partitions reproduced in <75% of bootstrap replicates are collapsed. The PLMVd genome sequences obtained in this study are marked with a red triangle.

Figure 6

Phylogenetic analysis of the identified viruses. The phylogenetic trees were constructed as for PLMVd in Fig. 5. The viral gene sequences obtained in this study are marked with a red triangle. (A) The nearly full genome and RdRp gene of NSPaV; (B) the CP and HSP70h genes of PBNSPaV; (C) the CP gene of ACLSV; (D) the CP gene of APV1.

Figure 7

Phylogenetic analysis of the identified viruses. The phylogenetic trees were constructed as for PLMVd in Fig. 5. The viral gene sequences obtained in this study are marked with a red triangle. (E) The CP gene of APV2; (F) the CP gene of CGRMV; (G) the CP gene of CNRMV; (H) the CP and RdRp genes of PaLV; (I) the CP gene of PeVD. NSPaV and barley yellow dwarf virus (BYDV) were used as outgroups in the PaLV tree. Grapevine rupestris vein feathering virus (GRVFV) and maize rayado fino marafivirus (MRFV) were used as outgroups in the PeVD tree.