Abstract
This thesis describes the results obtained during the three years PhD course which was focused on plant-bacteria interactions, in particular Vitis vinifera cultivar Glera and endophytes, previously isolated from the same plant species. The project aimed to investigate the possible beneficial effects that four selected endophyte strains, belonging to Pantoea and Bacillus genera, can give to Glera rooting cuttings and their potential utility as environmental-friendly alternative approach in plant growth promotion, stress alleviation, and biocontrol. The project was prompted to reduce the extensive use of agro-chemicals in agriculture and to promote a sustainable management of viticulture. As a first step, P. agglomerans (GL83) and B. licheniformis (GL174) selected for their valuable plant growth promoting traits were GFP-tagged and used to inoculate in vitro Glera apical cuttings in order to confirm their endophytic nature. Exploiting laser scanning confocal microscopy and a cultivation dependent method the colonization of stem endosphere of Glera apical cuttings after 20 and 30 days post inoculation (dpi) was demonstrated. The analysis was then extended at 45, 52 and 60 dpi in Glera cuttings which were in the meantime transferred in hydroponic non-sterile medium. Both strains were mostly visualized inside the xylem vessels of the stem until 60 dpi confirming the strains endophytic attitude and persistence. Then, the four strains were directly applied in the field by inoculating rootstocks (Kober 5BB) and Glera buds with each bacteria suspension (GL83, GL174, GL189, GL452 and the consortium of these four) both during the hydration step of rooting cuttings production line and by administering the bacterial suspensions at the root level, after plants have developed roots. Then, both the morphological and physiological plant parameters were recorded sampling plants at different time-point of their development. GL83 and GL174 were the most promising grapevine bio-fertilizer strains, especially when rootstocks and buds were inoculated during the hydration step of rooting cuttings production line. Inoculated plants presented improved leaf physiological parameters such as photosynthesis rate and transpiration flux linked to an increase of root and shoot fresh biomasses compared to the control plants. Moreover, an experiment aimed to simulate a fertilization procedure using 1-year-old Glera grapevine plants, which were bio-fertilized once a week for seven consecutive weeks with GL83 and GL174 confirmed these strains as promising bio-fertilizer candidates. Furthermore, GL83 was able to relieve the effects of drought stress in inoculated Glera rooting cuttings. In fact, stressed-inoculated plants presented enhanced physiological parameters compared to stressed-control plants and, in addition, GL83 plant growth promoting traits seemed to be water stress dependent. Another issue investigated during these three PhD years was the role of Bacillus cyclic lipopeptides (LPs) families as elicitors of plant defence responses, through induced systemic resistance (ISR) pathway. LPs are amphiphilic compounds produced by some beneficial bacterial strains freely living in the soil or undergoing mutualistic interactions with plants. The natural functions of LPs suggest their role in antagonism toward other microorganisms, like plant pathogens, and their involvement in motility and attachment to surfaces encouraging bacteria biofilm formation and development. So far, the mechanisms underlying the induced physiological responses in host plants, upon the exposure to these compounds remain unclear. Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families (surfactin, fengycin and iturin) both the early cell response and the later events like defence-gene expression were evaluated. Through Evans blue test no cell death increase was detected challenging Arabidopsis cell culture with three different concentrations of the LPs families. The administration of the highest concentration of surfactin (50 µg/ml) to A. thaliana cell cultures expressing the Ca2+-sensitive photoprotein aequorin in the cytosol evoked a cytosolic Ca2+ transient, suggesting that the perception of surfactin could be mediated by Ca2+, which is one of the main important intracellular messenger that generate a wide range of different spatial and temporal signals depending on the stimulus perceived. Then, to evaluate the possible long-term effects generated upon the perception of LPs, a semi-quantitative PCR defence-gene expression analysis was performed at 2 and 6 hours after LPs cell culture-exposure. Lipoxygenase 1 (LOX1), Pathogen-related protein 1 (PR1), Phenylalanine ammonia lyase (PAL1), Non-expressor of Pathogenesis-Related genes 1 (NPR1) and Mitogen-Activated Protein Kinase 3 (MAPK3) were the main genes investigated which are considered reliable markers of ISR and systemic acquired resistance (SAR). The results showed that ~3-fold increase of LOX1 and PAL1 expression was recorded in treated samples respect to controls. A ~2-fold increase in the NPR1 gene expression was observed comparing samples at 2 h with control. No differences in PR1 and MAPK3 gene expression were detected between samples and controls. The up-regulation of the LOX1 and NPR1 could suggest that surfactin elicits the activation of jasmonate-mediated pathway that could culminate in ISR. Instead, up-regulation of PAL1 without PR1 expression may indicate an initial SAR triggering, afterwards inhibited by some steps of ISR pathway. A UPLC-MS analysis performed at the Gembloux Agro-Bio Tech (University of Liège, Belgium) revealed that some selected Glera Bacillus endophytes produce surfactin and fengycin. Moreover, a quite uncommon type of surfactin called pumilacidin was detected and semi-purified. An in vitro antagonism assays against some plant pathogen bacteria showed that pumilacidin had a quite strong antibacterial effect. This is a preliminary result and other tests against, for example, infectious fungi such as B. cinerea need to be performed. Nevertheless, this is an important starting point for developing new sustainable practices with potential application in disease control. In conclusion, among grapevine cultivable endophytic strains, GL83 and GL174 showed to be promising candidates as bio-fertilizer enhancing both the grapevine growth and health: GL83 was able to promote growth and to confer an increased tolerance to drought stress whereas GL174 seemed to have a protecting action against some fungal grapevine pathogens. The possibility of endophyte exploitation to help plants to cope with abiotic as well as biotic environmental stresses, interfering with plant morphology and physiology or priming their systemic responses against pathogens, opens new scenarios for an environmentally friendly shift in agricultural practices with much lower impact of synthetic agro-chemicals. Results obtained from this work are aligned with the huge amount of studies, grown exponentially in the last years, in which the endophytes abilities and properties are often assayed in a single plant species or within groups of closely related plant genotypes. In addition, investigations are usually performed using microbial species that are relatively easy to cultivate leaving out that plant biome is always characterized by wide and complex interactions. The plant phenotype is determined not only by the plant responses to the environment but it is also orchestrated by the associated microbiota, the responses of the microbiota to the environment, and the complex interactions between individuals in the endosphere microbial plant community. Future exciting challenges, based on classical approaches and new valuable technologies (i.e. next-generation sequencing) applied to greenhouse and field conditions would allow to explore and characterize the contributions of genetic and metabolic elements involved in the interactions between host plants and endophytes, providing new ecological and evolutionary insights and a better knowledge of the plant-microbiome-environment relationships.
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Claims 16
Claim · model-written summary
Glera apical cuttings were colonized by the endophytes after 20 and 30 days post inoculation
- Taxon
- Vitis vinifera
Vitis viniferae0 - Material
- Glera apical cuttingse2
- Organ / stage
- stem endospheree2
- Interactor
- P. agglomerans (GL83)
Pantoea agglomeranse1symbiont - Second interactor
- B. licheniformis (GL174)
Bacillus licheniformise1symbiont - Outcome
- colonizatione2
- Qualifier
- after 20 and 30 days post inoculation (dpi)e2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e2“Exploiting laser scanning confocal microscopy and a cultivation dependent method the colonization of stem endosphere of Glera apical cuttings after 20 and 30 days post inoculation (dpi) was demonstrated”
e1“P. agglomerans (GL83) and B. licheniformis (GL174)”
Claim · model-written summary
Both endophyte strains were visualized inside the xylem vessels of the stem until 60 dpi
- Taxon
- Vitis viniferaVitis viniferae0
- Organ / stage
- xylem vessels of the steme3
- Interactor
- P. agglomerans (GL83)Pantoea agglomeranse1symbiont
- Second interactor
- B. licheniformis (GL174)Bacillus licheniformise1symbiont
- Outcome
- visualizede3
- Qualifier
- until 60 dpie3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e3“Both strains were mostly visualized inside the xylem vessels of the stem until 60 dpi confirming the strains endophytic attitude and persistence”
e1“P. agglomerans (GL83) and B. licheniformis (GL174)”
Claim · model-written summary
Inoculated grapevine plants had improved photosynthesis rate and transpiration flux compared with control plants
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- Inoculatede4
- Outcome
- photosynthesis rate and transpiration fluxe4
- Direction
- improvede4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e4“Inoculated plants presented improved leaf physiological parameters such as photosynthesis rate and transpiration flux linked to an increase of root and shoot fresh biomasses compared to the control plants”
Claim · model-written summary
Inoculated grapevine plants had an increase of root and shoot fresh biomasses compared with control plants
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- Inoculatede4
- Outcome
- root and shoot fresh biomassese4
- Direction
- increasee4
- Comparator
- control plantse4
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e4“Inoculated plants presented improved leaf physiological parameters such as photosynthesis rate and transpiration flux linked to an increase of root and shoot fresh biomasses compared to the control plants”
Claim · model-written summary
GL83 and GL174 were confirmed as promising grapevine bio-fertilizer candidates after weekly bio-fertilization for seven weeks
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- bio-fertilized once a week for seven consecutive weeks with GL83 and GL174e5
- Interactor
- GL83e5symbiont
- Second interactor
- GL174e5symbiont
- Outcome
- these strains as promising bio-fertilizer candidatese5
- Direction
- confirmede5
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e5“Moreover, an experiment aimed to simulate a fertilization procedure using 1-year-old Glera grapevine plants, which were bio-fertilized once a week for seven consecutive weeks with GL83 and GL174 confirmed these strains as promising bio-fertilizer candidates”
Claim · model-written summary
GL83 relieved the effects of drought stress in inoculated Glera rooting cuttings
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- drought stresse6
- Interactor
- GL83e6symbiont
- Outcome
- effects of drought stresse6
- Direction
- relievee6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e6“Furthermore, GL83 was able to relieve the effects of drought stress in inoculated Glera rooting cuttings”
Claim · model-written summary
Stressed inoculated grapevine plants had enhanced physiological parameters compared with stressed control plants
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- stressed-inoculated plantse7
- Interactor
- GL83e7symbiont
- Outcome
- physiological parameterse7
- Direction
- enhancede7
- Comparator
- stressed-control plantse7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e7“In fact, stressed-inoculated plants presented enhanced physiological parameters compared to stressed-control plants and, in addition, GL83 plant growth promoting traits seemed to be water stress dependent”
Claim · model-written summary
Surfactin-treated Arabidopsis cell cultures showed about 3-fold higher LOX1 and PAL1 expression than controls
- Taxon
- Arabidopsis
Arabidopsise2 - Treatment
- LPs cell culture-exposuree3
- Gene
- LOX1 and PAL1e1
- Outcome
- expressione1
- Direction
- increasee1
- Value
- ~3-folde1
- Comparator
- controlse1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“The results showed that ~3-fold increase of LOX1 and PAL1 expression was recorded in treated samples respect to controls”
e2“Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families”
e3“after LPs cell culture-exposure”
Claim · model-written summary
About 2-fold increase in NPR1 gene expression in Arabidopsis cell cultures at 2 h compared with control
- Taxon
- ArabidopsisArabidopsise1
- Treatment
- LPs cell culture-exposuree2
- Gene
- NPR1e3
- Outcome
- NPR1 gene expressione3
- Direction
- increasee3
- Value
- ~2-folde3
- Qualifier
- at 2 he3
- Comparator
- controle3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families”
e2“after LPs cell culture-exposure”
e3“A ~2-fold increase in the NPR1 gene expression was observed comparing samples at 2 h with control”
Claim · model-written summary
No differences in PR1 and MAPK3 gene expression between LPs-treated samples and controls
- Taxon
- ArabidopsisArabidopsise1
- Treatment
- LPs cell culture-exposuree2
- Gene
- PR1 and MAPK3e3
- Outcome
- PR1 and MAPK3 gene expressione3
- Direction
- No differencese3
- Comparator
- controlse3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Challenging Arabidopsis cell suspension cultures with the commercial Bacillus LPs families”
e2“after LPs cell culture-exposure”
e3“No differences in PR1 and MAPK3 gene expression were detected between samples and controls”
Claim · model-written summary
No cell death increase was detected in Arabidopsis cell cultures challenged with three concentrations of LPs families
- Taxon
- ArabidopsisArabidopsise1
- Treatment
- three different concentrations of the LPs familiese1
- Outcome
- cell deathe1
- Direction
- no cell death increasee1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“Through Evans blue test no cell death increase was detected challenging Arabidopsis cell culture with three different concentrations of the LPs families”
Claim · model-written summary
Surfactin evoked a cytosolic Ca2+ transient in Arabidopsis cell cultures
- Taxon
- A. thalianaArabidopsis thalianae2
- Treatment
- the highest concentration of surfactine1
- Outcome
- cytosolic Ca2+ transiente2
- Direction
- evokede2
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e1“The administration of the highest concentration of surfactin”
e2“to A. thaliana cell cultures expressing the Ca2+-sensitive photoprotein aequorin in the cytosol evoked a cytosolic Ca2+ transient”
Claim · model-written summary
Some selected grapevine Bacillus endophytes produce surfactin and fengycin
- Taxon
- Vitis viniferaVitis viniferae0
- Interactor
- Bacillus endophytese1symbiont
- Outcome
- produce surfactin and fengycine1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e1“some selected Glera Bacillus endophytes produce surfactin and fengycin”
Claim · model-written summary
Pumilacidin, a type of surfactin, was detected and semi-purified from the endophytes
- Taxon
- Vitis viniferaVitis viniferae0
- Outcome
- pumilacidin was detected and semi-purifiede1
- Qualifier
- a quite uncommon type of surfactine1
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e1“a quite uncommon type of surfactin called pumilacidin was detected and semi-purified”
Claim · model-written summary
Pumilacidin had a quite strong antibacterial effect against plant pathogen bacteria in vitro
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- pumilacidine1
- Interactor
- plant pathogen bacteriae1pathogen
- Outcome
- antibacterial effecte1
- Setting
- in vitroe1
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e1“An in vitro antagonism assays against some plant pathogen bacteria showed that pumilacidin had a quite strong antibacterial effect”
Claim · model-written summary
GL174 seemed to have a protecting action against some fungal grapevine pathogens
- Taxon
- Vitis viniferaVitis viniferae0
- Treatment
- GL174e1
- Interactor
- fungal grapevine pathogense1pathogen
- Outcome
- protecting actione1
Not reportedComparatorDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Vitis vinifera cultivar Glera”
e1“GL174 seemed to have a protecting action against some fungal grapevine pathogens”
The same questions in other papers
inoculation · physiology: photosynthesis · Vitis vinifera (wine grape)Positive and negative results
1 paper in the same direction as this paper · 1 in the opposite direction
- negative
- positive
Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.