Prevention and control of new and invasive geminiviruses infecting vegetables in the Mediterranean – GeMed

Methods and technologies used are presented according to the three main objectives of the project:
A. On the ecology side, standardized techniques will be used to assess the prevalence of geminiviruses in the Mediterranean Basin (MB) and according to host species: collection of plant and insect samples, storage, DNA extraction and viral detection tests using PCR and deep sequencing. Targeted geminiviruses are transmitted by the whitefly Bemisia tabaci, except Chickpea chlorotic dwarf virus (CpCDV) transmitted by leafhoppers. Whereas CpCDV was detected in the MB, the leafhopper vector was not detected. To spot the CpCDV leafhopper vector in the MB, we will use transmission tests, morphological observations, and molecular analysis.
B. To improve the understanding of the emergence of invasive geminiviruses, we will use IS76, a recombinant of Tomato yellow leaf curl virus that invaded Morocco and reached Spain. IS76 has emerged by positive selection exerted by the Ty-1 resistance gene of tomato cultivars. The genetic and molecular determinants of this positive selection will be investigated with engineered recombinants, transcriptomics, and small RNAomics. IS141 is an Italian TYLCV recombinant similar to IS76. Experiments performed in controlled conditions revealed that it is positively selected by Ty-1 resistant cultivars. Its invasive potential will be assessed with field surveys.
C. Standard techniques of integrated pest management (IPM) will be tested including mulching, intercropping and trapping of insect vectors. In parallel, essential oils will be tested for the control of insect vectors and new sources of resistance will be searched. Finally RNA vaccination will be tested for the control of geminiviruses.

A- Ecology of geminiviruses
Common protocols for sampling and processing plants and phloem-feeding insects have been approved. Their setting-up was possible following: (a) determination of the size of plant sample pools (b) preparation and shipping between partners of the standards required for virus screening, and (c) achievement of a ring test to standardize virus diagnostic procedures. For preservation, leaf samples were CaCl2-dried and insects were kept in ethanol.
To find the leafhopper vector of chickpea chlorotic dwarf virus (CpCDV), we had to get familiar with the complex morphological criteria used by taxonomists to distinguish leafhopper species. Based on microscopic observations of a large number of leafhoppers collected in Tunisia, a leafhopper species potentially vector of CpCDV was identified. Transmission tests were used as a parallel approach to spot leafhoppers vectors of CpCDV.
B- Understand outbreak phenomena of invasive recombinant geminiviruses
An RNAomic analysis was initiated to identify virus-plant interactions that are associated with resistance breaking phenomenon involving Ty-1 resistant tomato plants and invasive Tomato yellow leaf curl virus (TYLCV) recombinants. Libraries of small RNA and transcripts have been prepared and Illumina deep sequenced. The sequence analysis is in progress.
C- Diversify integrated pest management solutions
Citrus aurantium oil was extracted from the fruit peel and tested for whitefly control and toxicity to plants. Preliminary tests showed a toxic effect of the oil preparation on Bemisia tabaci. The 10% concentration is phytotoxic.
Several accessions of Cucurbita moschata were considered as donors of resistance traits to zucchini accessions regarding Tomato leaf curl New Delhi virus (ToLCNDV).
A strategy using a single dsRNA fragment targeting three regions of the A component of the ToLCNDV genome was tested. The dsRNA construct was prepared and preliminary tests are in progress in controlled conditions.

The project aims at providing stakeholders a distribution map of vegetable geminiviruses and their satellite DNA in the Mediterranean. As all the partners use the same sampling and virus detection protocols, the results are expected to provide an estimation of the relative prevalence of geminiviruses between countries. Besides the monitoring of previously reported geminiviruses in the Mediterranean, the project is also expected to test the possible occurrence of unreported geminiviruses potentially emerging in the Mediterranean. To do this, phloem-feeding insects collected in and around vegetable crops are used as samplers of plant viruses present in the environment. The virus genome of potential geminiviruses is amplified and deep sequenced.

The chickpea chlorotic dwarf disease is expected to be more efficiently controlled at the end of the project when its leafhopper vector and its ecology will be described.

A reconstruction of the emergence scenario of an invasive geminivirus in Morocco will be available. It is expected to increase the awareness of the risk of emergence of new variants that may compromise established control methods.

The project is expected to provide various experimental results that can be used by stakeholders to improve the control of geminiviruses. It includes the improvement of regular IPM tools with essential oils, intercropping, mulches, and yellow traps. Trials with topical applications of dsRNA or siRNA on plants will provide results on the efficiency and feasibility of RNA vaccination to control geminiviruses. The screening of accessions belonging to the Cucurbita genus for the resistance to ToLCNDV will potentially reveal new sources of resistance. Finally, intergenomic interactions between tomato and invasive TYLCV recombinants detected by an RNAome analysis are expected to reveal genes that may be exploited for breeding new TYLCV-resistant tomato cultivars.

No scientific production so far

Tomatoes and cucurbits are among the major vegetables grown in the Mediterranean, ranking 2nd and 3rd after potatoes. Their intensive production, with year round crops and a limited number of cultivars expose them permanently to the emergence and invasions of pathogens including viruses.

Geminiviruses are among the most worrying viruses of these crops due to their economic impact, the frequent introduction of new exotic species into the Mediterranean and the continuous emergence of potentially invasive and resistance breaking strains generated by recombination.

Prevention and control of these viruses is the major objective of GeMed project. It will be tackled by virologists, entomologists, geneticists, breeders, biologists and computer scientists. The specific objectives are within the major challenges of topic 1.2.2,
(i) broadening the knowledge of the ecology of new and potentially invasive geminiviruses with the involvement of partners located at the four cardinal points of the Mediterranean,
(ii) understanding outbreak phenomena of invasive recombinant geminiviruses with field observations and analysis of plant-virus interaction using resistance-breaking viral clones and deep small RNA-ome and transcriptome sequencing and bioinformatics
(iii) diversifying integrated pest management solutions against insect vectors with plant derived metabolites and against viruses with RNA vaccination of crop plants.

The innovation potential of GeMed is in the exploration of molecular mechanisms underpinning an outbreak phenomenon, the search of new resistance genes, and validation of exogenous RNAi based plant protection approaches.

Stakeholder knowledge and the potential of exploitation and dissemination of the result is embedded in the consortium with full participation of two seed companies and the association with various professional organisations interested in GeMed. Larger dissemination will be done via International plant protection organisations (EPPO, ProMED).