Date Published: January 17, 2019
Publisher: Public Library of Science
Author(s): Naama Levitzky, Elisheva Smith, Oded Lachman, Neta Luria, Yaniv Mizrahi, Helen Bakelman, Noa Sela, Orly Laskar, Elad Milrot, Aviv Dombrovsky, Graziella Berta.
The bumblebee Bombus terrestris is a beneficial pollinator extensively used in tomato production. Our hypothesis was that bumblebee hives collected from a Tomato brown rugose fruit virus (ToBRFV) infected tomato greenhouse, preserve an infectious primary inoculum. Placing a bumblebee hive collected from a ToBRFV contaminated greenhouse, in a glass-/net-house containing only uninfected healthy tomato plants, spread ToBRFV disease. Control uninfected tomato plants grown in a glass-/net-house devoid of any beehive remained uninfected. ToBRFV-contaminated hives carried infectious viral particles as demonstrated in a biological assay on laboratory test plants of virus extracted from hive components. Viral particles isolated from a contaminated hive had a typical tobamovirus morphology observed in transmission electron microscopy. Assembly of ToBRFV genome was achieved by next generation sequencing analysis of RNA adhering to the bumblebee body. Bumblebee dissection showed that ToBRFV was mostly present in the abdomen suggesting viral disease spread via buzz pollination. These results demonstrate that bumblebee hives collected from ToBRFV-contaminated greenhouses carry a primary inoculum that reflects the status of viruses in the growing area. This new mode of ToBRFV spread by pollinators opens an avenue for detection of viruses in a growing area through analysis of the pollinators, as well as emphasizes the need to reevaluate the appropriate disease management protocols.
Tomato production is jeopardized by the two newly discovered tobamoviruses Tomato mottle mosaic virus (ToMMV), reported in Brazil , Mexico  and USA , and Tomato brown rugose fruit virus (ToBRFV) reported in Jordan  and Israel .
A bumblebee from contaminated hive E was washed with 0.01M Phosphate buffer pH 7.0. The collected washing solution was analyzed for ToBRFV that adheres to the bee using RT-PCR followed by ToBRFV sequencing covering 5,267bp, and transcriptomic NGS (Illumina His-seq) (Fig 5). Bioinformatics analysis allowed to assemble the ToBRFV genome from the RNA extracted from the washing solution (Fig 5C) while no ToBRFV genome sequences were detected in the body extract of the washed bee. In addition, body parts of dissected bumblebees from infected and un-infected hives were analyzed for the presence of ToBRFV using RT-PCR (Fig 6B and 6C). The results obtained by the RT-PCR analysis showed positive detection of ToBRFV in the abdomens of bumblebees from contaminated hives (5/10), while the heads were negative for the virus (0/10).
Tobamoviruses are mechanically transmitted seed borne viruses that show very low seed transmission rate [20–23]. This suggests that fertilization process and embryonal contamination are not essential for tobamovirus spread. However, in a recent study we found that Cucumber green mottle mosaic virus (CGMMV) is spread from CGMMV-infected melon and cucumber plants to non-infected plants during honeybee Apis mellifera pollination in a greenhouse . It was therefore suggested that the tobamovirus physically adsorbs to the honeybees and the virus is mechanically spread while the honeybees are foraging . In tomato plants, bumblebees are more efficient than honeybees in their contribution to fruit production and quality  It has been reported that the bumblebee Bombus terrestris is able to transmit the TMV virus between infected and adjacently planted uninfected tomato plants . The experiments were carried out in greenhouses already containing a primary inoculum of TMV in the plants. Importantly, the TMV-contaminated bumblebees were not successful in transmitting the virus to healthy un-infected tomato plants grown in a greenhouse separate from TMV infected tomato plants . The data in that report implies that in the bumblebee hives there is no preservation of infectious TMV viral particles. We wanted to find out whether this conclusion is true for a greenhouse infected bumblebee hives. In our current study, we therefore employed a different strategy. We placed ToBRFV-contaminated-beehives, which were previously positioned in a virus-contaminated greenhouse, in our experimental glass-/net-houses containing only un-infected healthy tomato plants. In these designed experiments we prevented ToBRFV contamination via biting insects and small animals (e.g. mice). Primarily we asked whether infectious ToBRFV particles are preserved in the beehives and might constitute a primary inoculum transmitted by the bumblebees. In our designed experiments we are also avoiding any alterations in the bumblebee foraging behavior that may result from possible preference of the bumblebees to visit infected tomato plants versus the un-infected plants, as was observed in CMV-infected tomato plants . Under the latter circumstances, in a greenhouse that has both infected and un-infected tomato plants the bumblebees may revisit the virus infected plants and thereby reduce visits to the un-infected plants. Thus, the apparent virus spread might not reflect the full potential of the hive to disperse the virus. We therefore analyzed the spreading potential of ToBRFV-contaminated hives in glass-/net-houses containing only un-infected tomato plants. Indeed, we observed that the bumblebees that were inserted into the un-infected-glass-/net-house were transmitting ToBRFV (Fig 1 and Table 1). However, a disadvantage of our designed experiments that were based on greenhouse-contaminated-ToBRFV hives was the demonstration of high variation between contaminated hives, as reflected in the varied ratio of infected plants versus un-infected plants between experiments. This may be the result of bumblebee vitality/activity that was accordingly modified during the experiments. Importantly, the pattern of the infection was sporadic which is consistent with our care to prevent any mechanical transmission of the virus by any other means than via the bumblebees. This was also confirmed by the control experiments that were negative for ToBRFV.