Development of novel plant biofertilisers based on endophytic bacteria and innovative insect-mediated delivery strategies

As a result of the constant demand to ensure food security and crop growth, there is an urgent need of a more sustainable and resilient agriculture. Chemical fertilisers and pesticides are widely used in conventional agriculture and they cause possible environmental impacts. Tomato (Solanum lycopersicum) is cultivated worldwide under field and greenhouse conditions, requires an extensive use of chemical fertilisers and is greatly affected by arthropod feeding damages. Microbial biofertilisers and non-microbial biostimulants can contribute to plant development. Among them, plant growth promoting endophytic bacteria can internally colonise plant tissues and promote plant growth. Likewise, humic acids (HA) are organic substances that improve soil structure and can facilitate plant nutrient uptake. However, scarce information is available on the synergistic effects of endophytic bacterial strains and HA on the bacteria-mediated plant growth promotion. Moreover, beneficial insects, including the generalist predators Macrolophus pygmaeus and Nesidiocoris tenuis, can be used in biological control for the management of crop arthropods. Despite the significance of insects as vectors of plant pathogens is unquestionable, the transmission of beneficial microbes by beneficial insects is unknown. The aims of this thesis were to get insight into the molecular basis of the interaction between endophytic bacterial strains and tomato plants in the presence of HA, in order to improve the understanding on the mechanism responsible for plant growth stimulation, and to investigate the possible use of beneficial insects as vectors to transmit selected endophytic bacterial strains for the further development of sustainable delivery strategies of bacterial biofertilisers. In order to understand the complementation effects and cellular pathways activated by endophytic bacterial strains and HA, three bacterial strains (namely Paraburkholderia phytofirmans PsJN, Pantoea agglomerans D7G and Enterobacter sp. 32A) that were able to promote tomato shoot length in the presence of HA were selected. Double labelling of oligonucleotide probes for fluorescent in situ hybridisation was used to study tomato colonisation by endophytic bacterial strains. In particular, the colonisation intensity of tomato root and stem among the tested strains were comparable in the control and HA condition. Moreover, transcriptomics approach was applied to study the molecular mechanisms activated in tomato shoots and roots in response to endophytic bacteria and HA. Tomato genes were modulated by endophytic bacterial strains mainly in roots, indicating major transcriptional regulations in 3 belowground compared to aboveground tissues. The majority of DEGs was modulated by more than two strains, involving protein metabolism, transcription, transport, signal transduction and defence, representing possible common pathways modulated in response to bacterial endophytes. Moreover, strain-specific tomato responses involved signal transduction, transcription, hormone metabolism, protein metabolism, secondary metabolism and defence processes, highlighting specific traits of the endophyte-tomato interaction. In particular, the presence of HA enhanced the activation of signal transduction, hormone metabolism, transcription, protein metabolism, transport, defence and growth-related processes in response to P. phytofirmans PsJN, P. agglomerans D7G and Enterobacter sp. 32A inoculation, in terms of number of modulated genes and fold change values, indicating additive effects of bacterial endophytes and HA in plant growth promotion mechanisms. In relation to the possibility to deliver endophytes by using insects, beneficial mirids of tomato (namely M. pygmaeus and N. tenuis) were tested as potential vectors to transmit P. phytofirmans PsJN and Enterobacter sp. 32A between tomato plants. Mirids feeding on seedinoculated tomato plants were able to acquire the beneficial bacterial strains. In particular, after contact with bacterial-inoculated plants, P. phytofirmans PsJN and Enterobacter sp. 32A were detected on the majority of mirid epicuticle and inside surface-sterilised insects. Moreover, both tested mirids transmitted the selected bacterial strains between tomato plants and P. phytofirmans PsJN and Enterobacter sp. 32A could be re-isolated from tomato shoots after mirid-mediated transmission. Our study demonstrated that P. phytofirmans PsJN and Enterobacter sp. 32A can move within tomato plants, from shoots to roots after mirid-mediated transmission, indicating that M. pygmaeus and N. tenuis can acquire non-pytopathogenic microbes and the polyphagous and mobile nature of mirids could facilitate the transmission of beneficial endophytes among crops. In summary, the overall work provide in-depth knowledge on the HA-dependent enhancement of growth-related processes stimulated by endophytic bacterial strains and demonstrates the potential of beneficial mirids in transmission of beneficial bacteria and paved the way for the further development of efficient HA- and mirid-mediated strategies for plant biofertilisation and beneficial bacteria delivery.