Global Advances in Tomato Virome Research: Current Status and the Impact of High-Throughput Sequencing

Viruses cause a big fraction of economically important diseases in major crops, including tomato. In the past decade (2011–2020), many emerging or re-emerging tomato-infecting viruses were reported worldwide. In this period, 45 novel viral species were identified in tomato, 14 of which were discovered using high-throughput sequencing (HTS). In this review, we first discuss the role of HTS in these discoveries and its general impact on tomato virome research. We observed that the rate of tomato virus discovery is accelerating in the past few years due to the use of HTS. However, the extent of the post-discovery characterization of viruses is lagging behind and is greater for economically devastating viruses, such as the recently emerged tomato brown rugose fruit virus. Moreover, many known viruses still cause significant economic damages to tomato production. The review of databases and literature revealed at least 312 virus, satellite virus, or viroid species (in 22 families and 39 genera) associated with tomato, which is likely the highest number recorded for any plant. Among those, here, we summarize the current knowledge on the biology, global distribution, and epidemiology of the most important species. Increasing knowledge on tomato virome and employment of HTS to also study viromes of surrounding wild plants and environmental samples are bringing new insights into the understanding of epidemiology and ecology of tomato-infecting viruses and can, in the future, facilitate virus disease forecasting and prevention of virus disease outbreaks in tomato

Rapid molecular detection and transmission of bacterial leaf streak pathogen, Xanthomonas oryzae pv. oryzicola, in rice seeds

Xanthomonas oryzae pv. oryzicola (Xoc) is a seed-borne bacterial pathogen of rice. In this study, a rapid molecular detection method in seeds was developed to prevent unwanted movement of infected materials and greater yield loss. Crude DNA from ground seed extract supernatant was precipitated in ethanol and centrifuged. DNA pellets of homogenous subsamples were pooled and werepurified using SDS or a DNA extraction kit. Three pairs of primers previously designed on Xoc isolate BLS-256 genome sequence robustly amplify Xoc-specific DNA sequences. Primer T40 was found to efficiently detect Xoc but only in single primer reactions. While primers 3864 and 3866 can amplify Xoc DNA separately, however, their multiplex PCR reactions were unsuccessful, probably due to substrate competition in the reaction mixture. Optimized PCR mixture and conditions were able to detect as low as 10 cfu/mL Xoc cells and the direct extraction method and conditions were able to detect as low as 104 cfu/mL inoculated cells using primer T40. Lastly, Xoc transmission through seeds was observed only in plant samples which were heavily infected. Recovery from infection, at around 25-50% was also observed in some cultivars which also manifested good grain filling at 56 days after transplanting

Stomatal penetration and temporal dynamics of ingress of two fungal isolates associated with leaf spot disease of Jasmine (Jasminum sambac L.)

Jasmine (Jasminum sambac L.) is an ornamental crop grown in South and Southeast Asia for its flowers for garlands, tea and essential oil production. However, certain fungal foliar and floral diseases significantly reduce its yield. In this study, Colletotrichum sp.-like (cylindrical conidia) and Fusarium sp.- like (lunate conidia) isolates causing leaf spot disease were characterized based on the ingression process and fungal germination in planta. Four-day single-spore cultures in PDA of isolating C1 (Colletotrichum sp.-like) and F2 (Fusarium sp.-like) were obtained. Suspensions of 108 conidia mL-1 were made and sprayed onto young leaves of jasmine. Specimen collection was done at 2, 6, 12, 24, 36, 48, 60 and 72 h after inoculation (hai). Formalin-acetic acid was used to clear the tissues and fungal structures are selectively stained using lactophenol-acid fuschin. Average ingression sites (IS) were counted for each time point under 50X magnification, then IS per leaf area (cm2) was calculated. Fungal isolates remain at the surface of the leaf until 36 h. IS of C1 and F2 almost doubled at 48 hai. However, ingression sites declined at 72 hai for both pathogens, which either imply a progression of sub surface colonization or unsuccessful penetration. Leaf yellowing and a few spots were observed at 48 hai for F2 and at 60 hai for C1. More severe necrotic leaf spots with yellow halo (severity rating of 5) were seen in plants inoculated with Fusarium sp.-like isolated than in those inoculated with Colletotrichum sp.-like isolated. Lastly, at 48 to 60 hai, it was evident that the pathogen started to seek for stomata, which seemed to be the preferred penetration site for both fungal pathogens

Identification of wood Rot fungi in the historic Baker Memorial Hall at the University of the Philippines, Los Baños Campus

Deterioration in heritage wood structures caused by wood-decay fungi is a worldwide concern. In the University of the Philippines Los Baños, decay fungi from wooden canopies of the Baker Memorial Hall were identified and its degrading ability was evaluated. Specimen collection was conducted on degraded canopies made of Mayapis (Shorea palosapis) with signs of white or green fungal growth. Fungi associated with the rotting symptoms were isolated, purified, characterized and identified. Colonies of fungal isolates were fast-growing in malt extract agar (MEA), with colony diameter reaching 5.60 % 0.43 cm (WRF8) and 5.15 % 0.25 cm (WRF5) after a day. Mycelia of isolates were hyaline and rhizoidal on water agar. Isolate WRF5 produced green, raised colonies that imparted yellow pigmentation on potato dextrose agar (PDA) and MEA. Generally, colonies were raised, radially striated with green center and white margin on PDA. Average length and width of WRF5 conidia were 3.19 % 0.33 %m and 2.73 % 0.36 %m, respectively. Cross-referencing morphological and cultural data with related literatures and identification keys indicated the isolated fungi were Trichoderma viride (WRF1), T. crassum (WRF3), T. reesei (WRF5), Rhizopus sp. (WRF8) and Coniophora sp. Furthermore, inoculation of 108 T. reesei conidia/mL on clean Mayapis wood blocks resulted to profuse fungal growth, with average weight loss recorded at 8.81 % 0.79% at 8 wks and 10.53 % 0.88% at 14 wks which indicated a considerable but slow wood degradation

A genome-wide association study identifies Arabidopsis thaliana genes that contribute to differences in the outcome of infection with two Turnip mosaic potyvirus strains that differ in their evolutionary history and degree of host specialization

Viruses lie in a continuum between generalism and specialism depending on their ability to infect more or less hosts. While generalists are able to successfully infect a wide variety of hosts, specialists are limited to one or a few. Even though generalists seem to gain an advantage due to their wide host range, they usually pay a pleiotropic fitness cost within each host. On the contrary, a specialist has maximal fitness within its own host. A relevant yet poorly explored question is whether viruses differ in the way they interact with their hosts’ gene expression depending on their degree of specialization. Using a genome-wide association study approach, we have identified host genes whose expression depends on whether hosts were infected with more or less specialized viral strains. Four hundred fifty natural accessions of Arabidopsis thaliana were inoculated with Turnip mosaic potyvirus strains with different past evolutionary histories and that shown different degrees of specialization. Three disease-related traits were measured and associated with different sets of host genes for each strain. The genetic architectures of these traits differed among viral strains and, in the case of the more specialized virus, also varied along the duration of infection. While most of the mapped loci were strain specific, one shared locus was mapped for both strains, a disease-resistance TIR-NBS-LRR class protein. Likewise, only putative cysteine-rich receptor-like protein kinases were involved in all three traits. The impact on disease progress of 10 selected genes was validated by studying the infection phenotypes of loss-of-function mutant plants. Nine of these mutants have altered the disease progress and/or symptoms intensity between both strains. Compared to wild-type plants six had an effect on both viral strains, three had an effect only on the more specialized, and two were significant during infection with the less specialized.Work was funded by Spain’s Ministerio de Ciencia e Innovación-FEDER grant PID2019-103998GB-I00 and Generalitat Valenciana grants GRISOLIAP/2018/005 and PROMETEU2019/012 to SFE. RG was supported Ministerio de Ciencia e Innovación-FEDER contract BES-2016-077078. MPSR was supported by a Young Investigators fellowship from the Universitat de València International 0.7 Cooperation Program.Peer reviewe

Natural variation in Arabidopsis thaliana rosette area unveils new genes involved in plant development

Growth is a complex trait influenced by multiple genes that act at different moments during the development of an organism. This makes it difficult to spot its underlying genetic mechanisms. Since plant growth is intimately related to the effective leaf surface area (ELSA), identifying genes controlling this trait will shed light on our understanding of plant growth. To find new genes with a significant contribution to plant growth, here we used the natural variation in Arabidopsis thaliana to perform a genome-wide association study of ELSA. To do this, the projected rosette area of 710 worldwide distributed natural accessions was measured and analyzed using the genome-wide efficient mixed model association algorithm. From this analysis, ten genes were identified having SNPs with a significant association with ELSA. To validate the implication of these genes into A. thaliana growth, six of them were further studied by phenotyping knock-out mutant plants. It was observed that rem1.2, orc1a, ppd1, and mcm4 mutants showed different degrees of reduction in rosette size, thus confirming the role of these genes in plant growth. Our study identified genes already known to be involved in plant growth but also assigned this role, for the first time, to other genes.This work was supported by Spain’s Ministerio de Ciencia e Innovación-FEDER Grant PID2019-103998GB-I00 and by Generalitat Valenciana Grants GRISOLIA/2018/005 and PROMETEU2019/012 to S.F.E. R.G. was supported by Ministerio de Ciencia e Innovación-FEDER contract BES-2016-077078. M.P.S.R. was supported by a Young Investigators fellowship from the Universitat de València International 0.7 Cooperation Program.Peer reviewe

Virome analysis of irrigation water sources provides extensive insights into the diversity and distribution of plant viruses in agroecosystems

Plant viruses pose a significant threat to agriculture. Several are stable outside their hosts, can enter water bodies and remain infective for prolonged periods of time. Even though the quality of irrigation water is of increasing importance in the context of plant health, the presence of plant viruses in irrigation waters is understudied. In this study, we conducted a large-scale high-throughput sequencing (HTS)-based virome analysis of irrigation and surface water sources to obtain complete information about the abundance and diversity of plant viruses in such waters. We detected nucleic acids of plant viruses from 20 families, discovered several novel plant viruses from economically important taxa, like Tobamovirus and observed the influence of the water source on the present virome. By comparing viromes of water and surrounding plants, we observed presence of plant viruses in both compartments, especially in cases of large-scale outbreaks, such as that of tomato mosaic virus. Moreover, we demonstrated that water virome data can extensively inform us about the distribution and diversity of plant viruses for which only limited information is available from plants. Overall, the results of the study provided extensive insights into the virome of irrigation waters from the perspective of plant health. It also suggested that an HTS-based water virome surveillance system could be used to detect potential plant disease outbreaks and to survey the distribution and diversity of plant viruses in the ecosystem

Managing the deluge of newly discovered plant viruses and viroids

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties

Biological and genetic characterization of Physostegia chlorotic mottle virus in Europe based on host range, location, and time

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses