Detection of RNA viruses in Solanum quitoense by high-throughput sequencing (HTS) using total and double stranded RNA inputs
Introduction
Solanum quitoense Lam, also known as lulo or naranjilla, is a fruit shrub native to the northwestern corner of South America that produces a tomato-sized fruit popular in the preparation of fresh juice, fermented beverages, nectars, ice cream, and fruit concentrates [13,14]. The S. quitoense fruit has a sweet and acidic taste that is highly regarded for its pleasant aroma and flavor, and is rich in fiber, citric acid, calcium, phosphorus, niacin, thiamin, riboflavin, vitamins A and C, and antioxidants [1,16,24,57]. All these properties have made S. quitoense an attractive exotic fruit with increased demand in the international market [11]. With an annual yield of about 89,050 t, Colombia is one of the top worldwide producers of lulo, which is mostly cultivated by small farmers concentrated in the provinces of Huila (2005 ha), Valle del Cauca (1029 ha), Cauca (702 ha), Nariño (608 ha), and Antioquia (600 ha) [2]. Currently, Antioquia is the first producer with the highest yields approximately 16.74 t/ha, doubling the yields of most the inefficient producers, such as Huila and Nariño, estimated at 8 t/ha [2].
The growing demand for exotic tropical fruits has incentivized lulo farmers to increase yields and cultivated area but, unfortunately, S. quitoense is highly susceptible to several pathogens and pests that limit its geographical expansion [31]. Some of these problems include insects such as the small tomato borer (Neoleucinodes elegantalis) and the stem-base borer (Faustinus sp.) [52]; the root-knot nematode Meloidogyne spp. [46]; the oomycete Phytophthora infestans [35]; fungi such as Colletotrichum gloeosporioides and Fusarium oxysporum [9,35], the soil-borne bacteria Ralstonia solanacearum [9,52], and a wide spectrum of viral diseases [15,20,52]. Until recently, there were no detailed reports of the viruses infecting S. quitoense naturally. Before 2018, studies of viruses in lulo crops were scant and mostly focused on the description of symptoms or the hypersensitive response to viruses affecting other solanaceous crops [23,52]. However, some studies on the S. quitoense virome have started to emerge in recent years. In 2018, a study in the Pastaza province of Ecuador revealed the existence of Naranjilla chlorotic mosaic virus (NarCMV), a new putative tymovirus species causing stunting and chlorotic mosaic symptoms [20]. That same year, a high-throughput sequencing (HTS) study reported the complete genome sequences of Cucumber mosaic virus (CMV), Potato yellow vein virus (PYVV), and Alstroemeria necrotic streak virus (ANSV) naturally infecting commercial S. quitoense crops in Colombia [15]. These advances were possible thanks to the use of HTS methods, which allow the identification of viruses without requiring specific antibodies and/or nucleic acid primers [33,59] allowing the determination of the plant viromes.
High-throughput sequencing are going to be a standard method in the diagnosis of viruses, and it is likely that they will be routinely used as part of the quarantine decision-making process [32,33]. Unfortunately, viral sequences can be present at low abundance in a background of highly abundant plant sequences. In the case of RNA sequencing, the virus signal can be improved by selectively removing plant sequences using techniques such as subtractive hybridization and ribosomal RNA depletion, or enriched using purification methods for viral particles, small interfering RNA (siRNA), polyadenylated RNA, and double stranded RNA (dsRNA) [40,44]. For example, an HTS sequencing study on Syrah grapevines with decline symptoms using total RNA and dsRNA found that viral reads increased from 2% to 53% after dsRNA enrichment [3]. As part of an initiative to characterize the viruses infecting S. quitoense in Colombia, we have tested the effect of using double stranded RNA (dsRNA) and ribosomal RNA depletion methods on the detection of viruses in bulked leaf samples with severe symptoms of viral infection from eastern Antioquia (Colombia).
Section snippets
Plant material
Sequencing was performed on bulked leaf samples collected at six plots from the municipalities of Marinilla (6° 10′ 24.89″ N, −75° 20′ 10.36″ W) and La Unión (5° 58′ 14.99″ N, −75° 21′ 24.59″ W) in the province of Antioquia (Colombia). Leaf samples exhibited symptoms typical of viral infections such as green vein banding, interveinal chlorosis and leaf chlorosis, severe leaf deformation, rugose mosaics and enation (Supplementary Fig. 1).
RNA extraction
Extractions were performed on samples ground in liquid
Data composition
A total of 31,825,448 reads (3,214,370,248 total bases) and 52,486,564 reads (5,301,142,964 total bases) were obtained from the total RNA and dsRNA libraries, respectively (Supplementary Fig. 3). The total RNA data could be reduced into a set of 11,012,971 of non-redundant sequences (34.6%); 68.9% of reads were unique, 29.0% were repeated between 2 and 10 times, and 2.0% were repeated more than 10-fold. In contrast, the dsRNA dataset could be reduced into a set of 4,110,433 (7.8%) unique
Discussion
HTS has become one of the most promising techniques for the diagnosis of viruses in integrated disease management programs. This methodology has facilitated the identification and characterization of viruses with a speed and sensitivity unthinkable with older techniques. This work confirms the usefulness of high-throughput sequencing methods in the characterization of viromes of exotic crops. A previous study using HTS revealed that lulo can be naturally infected by PYVV, CMV and ANSV in
Conclusion
We conclude that both approaches are complementary as total RNA gives higher coverage for viruses present at high levels but dsRNA allows detection of viruses present at low titers and persistent viruses, as observed in other studies for members of the Partitiviridae, Endornaviridae and Totiviridae families [44]. We recommend using at least two different nucleic acids extraction methods for the study of plant viromes. Our work also reveals the high diversity of viruses infecting S. quitoense
Author contributions
YG, MM and PG designed the study and were involved in conducting experiments. YG and PG analyzed the results. YG, MM and PG prepared the manuscript. All authors read and approved the final version.
Ethical approval
This article does not contain any research involving human or animal participants.
Funding
This work was supported by Universidad Nacional de Colombia (Grant: 40817).
CRediT authorship contribution statement
Yuliana Gallo: Formal analysis. Mauricio Marín: designed the study and were involved in conducting experiments, prepared the manuscript. Pablo Gutiérrez: Formal analysis.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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