During the search for alternatives to the use of chemical pesticides and fertilizers, the use of microbial inoculants has emerged as a promising option. Soil microorganisms such as the arbuscular mycorrhizal fungi (AMF) and the entomopathogenic fungi (EPF) have shown beneficial effects on plant health and protection. While the AMF has an obligate symbiosis with plants, the EPF are mainly insect pathogens, but many EPF can also establish association with plants and this role during the interaction between EPF and plants is receiving increased attention.
So far, most studies have evaluated the effect of individual microbial inoculants on plant defense. This PhD work focused on the evaluation of plant defense induction when EPF and AMF were root-inoculated individually and in combination. Another approached followed in this PhD study was the selection of EPF isolates based on results obtained in greenhouse experiments to compare and evaluate the consistency of the effect obtained under commercial conditions.
In the present PhD study, the role of EPF in plant protection was first explored by root-inoculating tomato plants different isolates of Beauveria, Metarhizium and Isaria, which were then challenged by the aboveground insect herbivore Spodoptera exigua. Results related with the plant growth and effects on S. exigua were considered to select EPF isolates for the next experiments (Manuscript 1). In these, the EPF isolates B. bassiana KVL 13-39, M. brunneum KVL 16-36 and M. robertsii KVL 12-35 were combined with the AMF Funneliformis mosseae to evaluate the effects on plant defense against the foliar phytopathogen Botrytis cinerea (Manuscript 2). Next, the AMF Rhizophagus irregularis was combined with the same EPF isolates to test the effects on tomato plant defense against S. exigua and to evaluate the production of the specialized metabolites α-tomatine and dehydrotomatine. In addition, a targeted analysis of expression of defense genes was performed to evaluate the possible implication of signaling pathways for defense regulation (Manuscript 3). Finally, the effects of selected EPF, AMF and their combinations on plant defense and growth under realistic crop production conditions were evaluated (Annexes).
The EPF isolates showed differences in the persistence in tomato root systems (Manuscript 1) and plant-mediated effects on protection against the insect herbivores (Manuscript 1; Annexes) and a phytopathogen (Manuscript 2).
Some EPF isolates increased the biomass of tomato plants when inoculated individually (Manuscript 1) or in dual root inoculation with AMF (Manuscript 2, 3). Even though the presence of AMF in the roots decreased the EPF root colonization densities, some combinations such as F. mosseae with M. brunneum caused a significant increase in root biomass and F. mossease with M. brunneum, M. robertssii and B. bassiaana increased shoot biomass (Manuscript 2). While in Manuscript 3, the AMF did not affect the persistence of EPF in the roots, and the shoot biomass was increased by the same isolates of EPF while in the roots, the combination of M. brunneum and M. robertsii with the AMF Rhizophagus irregularis compensated the negative effect of insect herbivory. Nevertheless, for the studies performed in this work (Manuscripts 1, 2, 3) when experiments were repeated in time, some differences in results were found between the repetitions.
For induced defense against the phytopathogen B. cinerea, the three EPF isolates reduced the growth of the pathogen lesion on tomato leaves. In contrast, the AMF F. mosseae had no effect on B. cinerea lesions, while AMF showed positive effects on plant biomass (Manuscript 2). Contrary to expected, the three EPF isolates did not show any effect on the leaf concentration of the steroidal glucoalkaloids α-tomatine and dehydrotomatine, which are central defense compounds against herbivores in tomato. However, there were some changes in the expression pattern of defense genes related with hormonal signaling pathways. Opposite to the expectation, the presence of fungal inoculants, in particular AMF, did not increase the expression of the defense genes in leaves after being challenged to the insect herbivore S. exigua (Manuscript 3).
In the field experiment under commercial production conditions, it was found that although no defense effects against B. cinerea and S. exigua in bioassays with detached leaves were observed, a reduction in the natural incidence of pests on inoculated plants were recorded in the greenhouse at commercial scale.
Overall, the entomopathogenic fungi have the ability to induce plant defenses and affect plant growth when are associated with tomato plant roots. Furthermore, EPF and AMF have not stronger effects when combined, but the fungi have complementary functional roles related to plant defense and plant growth. In this work, the induction of systemic resistance at molecular level by the EPF when single and coinoculated with AMF was not observed.Therefore, the mechanisms of plant defense related to the induction of genes and metabolites during fungal interactions need to be further elucidated. Nevertheless, the effects of EPF on tomato plants were isolate dependent, and the results varied between the experimental repetitions for the trials performed, suggesting a high degree of context-dependency in these plant-microbe interactions. Such variable results may comprise a constraint for the future uptake and use of beneficial fungi in plant production.