Tomato (Solanum lycopersicum) is the second most important non-cereal crop worldwide and an important model species to study fruit physiology and development, quantitative genetics, and plant breeding. It is a major source of minerals, vitamins, and different antioxidants but its production is impaired by many biotic and abiotic factors. The major factors hindering tomato production are drought stress and insect pest infestation. Growers have used a wide array of pesticides and other chemical inputs to obtain good yields and decrease the loss. The use of environmentally friendly approaches to minimize chemical inputs is the goal of sustainable agriculture. Beneficial microorganisms associated to tomato may contribute sustainable plant growth promotion and stress tolerance, but tomato has not been sufficiently valorized as a plant-microbe interaction crop. Overall, this thesis focused on providing insights into the ability of plant beneficial strains belonging to the genus Pseudomonas to promote tomato growth and development, drought stress resilience and induced systemic effects against insect pests.
The experimental work for this thesis was organized in three main work packages. The first work package included screening for the effects on plant physiology of a panel of six plant beneficial Pseudomonas strains. The effects on plant physiology was determined as activity profiles of primary carbohydrate and antioxidant metabolism enzymes after root inoculation. The second work package investigated the role of cytokinin producing (Pseudomonas fluorescens G20-18) and biofilm forming (Pseudomonas putida KT2440) strains in protecting the tomato against drought stress. These studies included mutant strains, which were deficient in the above-mentioned characters to get insight into the bacterial properties of importance for protection against drought stress. The third work package was designed to investigate the potential of the above mentioned two bacterial strains for inducing systemic effects protecting against the leaf chewing insect beet armyworm (Spodoptera exigua).
The outcomes of this thesis are separated in to five manuscripts: one review and four research articles. Manuscript I reviewed the role of cytokinin (CK) in plant protection. The review emphasized that CKs are not only produced by plants, but also produced by bacteria, fungi, microalgae and insects. As this phytohormone has been known for its role in plant growth and development, we tried to assess its additional role for plant protection against bacterial and fungal pathogen and insect pests. We also highlighted its crosstalk with other phytohormones in plant growth and enhancing plant immunity against pathogen infections. Overall, the review pointed to a biocontrol ability of microbial derived CKs, which opens promising applications in integrated plant protection.
The objective of Manuscript II was to determine the impact of six different plant beneficial bacteria on tomato growth and the activity of specific enzymes involved in carbohydrate or antioxidant metabolism (metabolic biosignatures) after root inoculation. Moreover, we aimed at exploring if the different Pseudomonas strains cause similar or different metabolic biosignatures, and if any of the biosignatures are correlated with growth promotion. All the bacterial strains induced plant growth measured as biomass and plant height. They also increased leaf chlorophyll content and caused distinct carbohydrate and antioxidative metabolism enzyme activity profiles in leaf and root tissue, respectively. Each bacterial strain gave a very distinct metabolic biosignature and even there was correlation between some enzyme activities and the growth parameters measured. The increased activities of carbohydrate and antioxidant metabolism enzymes due to the inoculation could be relevant for growth and development but also for abiotic and biotic stress responses. The distinct metabolic biosignatures in tomato leaf and root tissue might be used as predictive markers for future selection and application of plant beneficial Pseudomonas strains. This is because screening for growth associated metabolic biosignatures would reduce time and cost for extensive screening for plant beneficial bacteria. These findings open new perspectives to integrate determination of metabolic biosignatures in screening of microbes at early developmental stages of the host plant.
In Manuscript III the objective was to explore the potential of the CK-producing bacterium P. fluorescens G20-18 to promote growth and enhance drought stress tolerance of tomato. An additional aim was, to reveal the specific role of bacterial produced CK in these processes by comparing the wild type with two TnphoA-derived mutants (CNT1 and CNT2) showing a reduced capacity to produce CK. According to the physiological analysis, drought stressed tomato plants inoculated with G20-18 and the CNT mutants had increased chlorophyll content, decreased stomatal conductance and increased leaf ABA content. Beyond growth promotion the strains also increased the activity of several carbohydrate and antioxidant metabolism enzymes, increasing total antioxidant capacity and accumulation of secondary metabolites during drought stress. As seen from gene expression analysis fivefold of genes were upregulated in G20-18 inoculated drought stressed plants compared with the number of genes upregulated before the start of the drought stress and during drought stress recovery. Major differences was seen in the number genes regulated by G20-18 and its CK deficient mutant CNT1 under well-watered and drought stress conditions indicating that the ability of G20-18 to produce this plant hormone is contributing to its beneficial effects both under non-stressed and stressed conditions. G20-18 and the CNT mutant strains induced comparable physiological responses but different metabolic and gene expression changes. This could indicate that the CKs had an impact on plant cell physiology and role in protection against drought stress.
The objective in Manuscript IV was to explore the potential of biofilm forming P. putida KT2440 to enhance drought stress tolerance of tomato. Additionally, the aim was to reveal the specific role of the biofilm by comparing the wild type with the two mutants (KT2440 Alg and KT2440 Q). In KT2440 Alg, genes encoding for exopolysaccharide alginate were deleted, whereas in KT2440 Q four identifiable putative gene clusters (including alginate and cellulose) were deleted. TheAlg and Q mutant strains show reduced or abolished capacity for biofilm formation in vitro, respectively. Inoculation with KT2440 resulted in increasing leaf water potential, having higher stomatal opening and higher photosynthetic rate in stressed plants. Inoculation with KT2440 Alg gave comparable results to inoculation with the wild type. In contrast, the effect of KT2440 Q was not different from that seen in non-inoculated plants. KT2440 also increased the activity of enzymes involved in resource allocation, assimilate partitioning and antioxidant metabolism. From the physiological measurements, KT2240 showed a direct impact on the examined tomato plants ability to retain water under drought stress. A difference in biofilm formation by the wild type KT2440 and, in particular the KT2440 Q mutant might be related to plant’s drought stress resilience, but more studies of biofilm formation on the roots is needed to validate this assumption.
Finally, Manuscript V focused on investigating the role of two bacterial strains P. fluorescens G20-18 and P. putida KT2440 on inducing systemic effect against insect attack on tomato. G20-18 inoculation led to plant protection with reduction of larval weight gain of the generalist Spodoptera exigua. G20-18 also significantly increased the activities of enzymes involved in carbohydrate partitioning (PFK, UGPase and PGI) and antioxidant defense (GR, MDHAR and GST). Comparing the effects of G20-18’s and KT2440, the latter strain showed less effect in plant protection and enzyme activities. Those enzymes where their activity was increased will bring the energy required during defense and could help in removal of ROS formed during insect feeding. The ability of this strain to induce systemic resistance could be further revealed by phytohormone profiling and analysis of gene expressions. The results from these analyses are pending.
In conclusion, the research performed in this Ph.D. thesis provides new insights into the interactions between beneficial Pseudomonas and tomato in the presence of biotic and abiotic stresses. Findings from this thesis can lead to future works in both basic and applied science and to new applications of these strains in plant protection research, based on their role in promoting growth and stress resilience of tomato.