The invasion biology of tomato begomoviruses in Costa Rica reveals neutral synergism that may lead to increased disease pressure and economic loss.

Since the late 1980s, tomato production in Costa Rica has been affected by diseases caused by whitefly-transmitted begomoviruses. The first was tomato yellow mottle virus (ToYMoV), a locally evolved New World (NW) bipartite begomovirus associated with the tomato yellow mottle disease (ToYMoD). In the late 1990s, the invasive NW bipartite tomato leaf curl Sinaloa virus (ToLCSiV) was detected in Costa Rica and has become established and associated with ToYMoD. Finally, the invasive Old World (OW) monopartite tomato yellow leaf curl virus (TYLCV) was detected in Costa Rica in 2012 and has also become established and is causing tomato yellow leaf curl disease (TYLCD). In the present study, we investigated the invasion biology of these tomato-infecting begomoviruses in Costa Rica in terms of (i) their biological and genetic properties and (ii) disease symptoms and viral DNA accumulation in tomato plants having single and mixed infections. We first generated infectious DNA-A and DNA-B clones and agroinoculation systems for ToYMoV and ToLCSiV isolates recovered from archival ToYMoD samples collected in Costa Rica in 1990 and 2002, respectively. Tomato plants agroinoculated with the infectious clones of both viruses developed ToYMoD symptoms, completing Koch's postulates for ToYMoV, and showing that ToLCSiV also causes this disease. However, pseudorecombinants formed between the DNA components of these viruses were not infectious, which is consistent with independent evolution in different lineages and limits genetic interactions. Furthermore, ToYMoV is well-adapted to tomato, has a narrow host range and is mechanically transmissible. The DNA-A component has a recombination event in the hot spot area and induced a symptomless infection in agroinoculated Nicotiana benthamiana and tomato plants. Tomato plants co-infected with two or all three viruses developed more severe symptoms compared with plants infected with each virus alone. Symptoms induced by the NW bipartite ToYMoV and ToLCSiV appeared earlier (∼7 d post-inoculation [dpi]) than those induced by TYLCV (∼10 dpi), but TYLCD symptoms became predominant in single and mixed infections by 14 dpi. Viral DNA accumulation was quantified by qPCR and generally revealed a neutral synergistic interaction in which the viruses co-existed in mixed infections. A transient reduction in accumulation of ToYMoV and ToLCSiV was detected in mixed infections at 7 dpi, whereas TYLCV accumulation was not affected in mixed infections and was uniform among treatments and time points. Together our results suggest that this neutral synergistic interaction will lead to increased begomovirus disease severity in Costa Rica. We discuss this in terms of begomovirus invasion biology and disease management.

Kinship networks of seed exchange shape spatial patterns of plant virus diversity.

By structuring farmers' informal networks of seed exchange, kinship systems play a key role in the dynamics of crop genetic diversity in smallholder farming systems. However, because many crop diseases are propagated through infected germplasm, local seed systems can also facilitate the dissemination of seedborne pathogens. Here, we investigate how the interplay of kinship systems and local networks of germplasm exchange influences the metapopulation dynamics of viruses responsible for the cassava mosaic disease (CMD), a major threat to food security in Africa. Combining anthropological, genetic and plant epidemiological data, we analyzed the genetic structure of local populations of the African cassava mosaic virus (ACMV), one of the main causal agents of CMD. Results reveal contrasted patterns of viral diversity in patrilineal and matrilineal communities, consistent with local modes of seed exchange. Our results demonstrate that plant virus ecosystems have also a cultural component and that social factors that shape regional seed exchange networks influence the genetic structure of plant virus populations.

Cassava Mosaic and Brown Streak Diseases: Current Perspectives and Beyond.

Cassava is the fourth largest source of calories in the world but is subject to economically important yield losses due to viral diseases, including cassava brown streak disease and cassava mosaic disease. Cassava mosaic disease occurs in sub-Saharan Africa and the Asian subcontinent and is associated with nine begomovirus species, whereas cassava brown streak disease has to date been reported only in sub-Saharan Africa and is caused by two distinct ipomovirus species. We present an overview of key milestones and their significance in the understanding and characterization of these two major diseases as well as their associated viruses and whitefly vector. New biotechnologies offer a wide range of opportunities to reduce virus-associated yield losses in cassava for farmers and can additionally enable the exploitation of this valuable crop for industrial purposes. This review explores established and new technologies for genetic manipulation to achieve desired traits such as virus resistance.

Evaluation of recombinase polymerase amplification for detection of begomoviruses by plant diagnostic clinics.

BACKGROUND: Plant viruses in the genus Begomovirus, family Geminiviridae often cause substantial crop losses. These viruses have been emerging in many locations throughout the tropics and subtropics. Like many plant viruses, they are often not recognized by plant diagnostic clinics due in large part to the lack of rapid and cost effective assays. An isothermal amplification assay, Recombinase polymerase amplification (RPA), was evaluated for its ability to detect three begomoviruses and for its suitability for use in plant diagnostic clinics. Methods for DNA extraction and separation of amplicons from proteins used in the assay were modified and compared to RPA manufacturer's protocols. The modified RPA assays were compared to PCR assays for sensitivity, use in downstream applications, cost, and speed. RESULTS: Recombinase polymerase amplification (RPA) assays for the detection of Bean golden yellow mosaic virus, Tomato mottle virus and Tomato yellow leaf curl virus (TYLCV) were specific, only amplifying the target viruses in three different host species. RPA was able to detect the target virus when the template was in a crude extract generated using a simple inexpensive extraction method, while PCR was not. Separation of RPA-generated amplicons from DNA-binding proteins could be accomplished by several methods, all of which were faster and less expensive than that recommended by the manufacturer. Use of these modifications resulted in an RPA assay that was faster than PCR but with a similar reagent cost. This modified RPA was the more cost effective assay when labor is added to the cost since RPA can be performed much faster than PCR. RPA had a sensitivity approximate to that of ELISA when crude extract was used as template. RPA-generated amplicons could be used in downstream applications (TA cloning, digestion with a restriction endonuclease, direct sequencing) similar to PCR but unlike some other isothermal reactions. CONCLUSIONS: RPA could prove useful for the cost effective detection of plant viruses by plant diagnostic clinics. It can be performed in one hour or less with a reagent cost similar to that of PCR but with a lower labor cost, and with an acceptable level of sensitivity and specificity.

Assessment of the genetic diversity of tomato yellow leaf curl virus.

The objective of the present study was to analyze the genetic diversity of tomato yellow leaf curl virus (TYLCV). Representative TYLCV sequences were searched in the National Center for Biotechnology Information database. Comprehensive analysis of TYLCV was performed using bioinformatics by examining gene structure, sequence alignments, phylogeny, GC content, and homology. Forty-eight representative TYLCV sequences were selected from 48 regions in 29 countries. The results showed that all TYLCV sequences were 2752-2794 nucleotides in length, which encoded 6 open reading frames (AV1, AV2, AC1, AC2, AC3, and AC4). GC content ranged from 0.41-0.42. Sequence alignment showed a number of insertions and deletions within these TYLCV sequences. Phylogenetic tree results revealed that the sequences were divided into 10 classes; homology of the sequences ranged from 72.8 to 98.6%. All 48 sequences contained the typical structure of TYLCV, including open reading frames and intergenic regions. These results provide a theoretical basis for the identification and evolution of the virus in the future.

Detoxification activity and energy cost is attenuated in whiteflies feeding on tomato yellow leaf curl China virus-infected tobacco plants.

The begomovirus Tomato yellow leaf curl China virus (TYLCCNV) can benefit its vector, the whitefly Bemisia tabaci, through suppressing the defences of their shared host plants. However, the mechanisms of this vector-virus mutualism remain largely unknown on the insect side of the interaction. Here, we compared the transcriptional profiles of female adult whiteflies of B. tabaci Middle East-Asia Minor 1 feeding on TYLCCNV-free and TYLCCNV-infected tobacco plants using the next-generation sequencing technique and quantitative real-time PCR. Interestingly, the genes involved in the oxidative phosphorylation (OXPHOS) pathway and detoxification enzyme were down-regulated in whiteflies feeding on virus-infected plants. Decreased detoxification activity costs less energy, which may reduce OXPHOS activity. Moreover, the genes involved in redox activity were also down-regulated, which may indicate that the reduced OXPHOS activity decreased reactive oxygen species production. Reduced detoxification activity is likely to attenuate energy costs, thereby enhancing the performance of whiteflies on virus-infected plants. These results provide further insight into the mechanisms of the plant-mediated whitefly-virus mutualism. Moreover, our study suggests that investigating the transcriptional profiles on the insect side of the interaction can advance our understanding of the tripartite interactions.

Safety assessment of leaf curl virus resistant tomato developed using viral derived sequences.

Genetic engineering of food crops has significantly influenced the agricultural productivity over the past two decades. It has proved a valuable tool, offering crops with higher yields, improved nutritional quality, resistance against pesticides, herbicides and tolerance against abiotic stresses. However, the safety assessment of genetically engineered (GE) crops is prerequisite before introduction into human food chain. The present study was aimed to assess the toxicity and allergenicity of leaf curl virus resistant GE tomato compared to its wild-type species. Balb/c mice fed with genetically engineered or wild-type tomato did not show significant differences in growth, body weight (P > 0.05) and food consumption when compared with control mice. Values for serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase, urea and cholesterol were comparable in GE and wild-type tomato fed mice. Mice immunized with GE or wild-type tomato extract showed low IgE response. Lung histology of ovalbumin fed mice showed bronchoconstriction with eosinophilic infiltration whereas GE or wild-type tomato showed no cellular infiltration with normal airways. Genetically engineered and wild-type tomato sensitized mice demonstrated similar IL-4 release in splenic cell culture supernatant. GE and wild tomato extract on ELISA showed comparable IgE binding (P > 0.05) with food allergic patients' sera. In conclusion, genetically engineered tomato showed no toxicity in mice and allergenicity is similar to the wild-type tomato.