Academic literature on the topic 'Endophytes plant growth-promoting bacteria (ePGPB)'

Endophytic plant-growth-promoting bacteria (ePGPB) are interesting tools for pest management strategies. However, the molecular interactions underlying specific biocontrol effects, particularly against phytopathogenic viruses, remain unexplored. Herein, we investigated the antiviral effects and triggers of induced systemic resistance mediated by four ePGPB (Paraburkholderia fungorum strain R8, Paenibacillus pasadenensis strain R16, Pantoea agglomerans strain 255-7, and Pseudomonas syringae strain 260-02) against four viruses (Cymbidium Ring Spot Virus—CymRSV; Cucumber Mosaic Virus—CMV; Potato Virus X—PVX; and Potato Virus Y—PVY) on Nicotiana benthamiana plants under controlled conditions and compared them with a chitosan-based resistance inducer product. Our studies indicated that ePGPB- and chitosan-treated plants presented well-defined biocontrol efficacy against CymRSV and CMV, unlike PVX and PVY. They exhibited significant reductions in symptom severity while promoting plant height compared to nontreated, virus-infected controls. However, these phenotypic traits showed no association with relative virus quantification. Moreover, the tested defense-related genes (Enhanced Disease Susceptibility-1 (EDS1), Non-expressor of Pathogenesis-related genes-1 (NPR1), and Pathogenesis-related protein-2B (PR2B)) implied the involvement of a salicylic-acid-related defense pathway triggered by EDS1 gene upregulation.

There is an increasing interest in the use of beneficial microorganisms as alternatives to chemically synthesized or plant-derived molecules to produce therapeutic agents. Bacterial endophytes are plant-associated microorganisms that can colonize different parts of living plants without causing any diseases. Diverse endophytic bacteria possess the ability to synthesize a wide range of secondary metabolites with unique chemical structures that have been exploited for their anti-microbial, antiviral, anti-cancer, and anti-inflammatory properties. Additionally, production of these bioactive compounds can also benefit the host plant as they may play a significant role in a plant’s interaction with the environment for adaptation and defense. As a result of their significant impact as curative compounds or as precursors to produce new drugs, the biotechnological possibilities of secondary metabolites derived from endophytic bacteria are immense.

The present study was carried out for the isolation and characterization of endophytic bacteria from chickpea nodules. A total of 107 endophytes were isolated from five districts of Haryana using three media, viz. YEMA for rhizobia, Pikovskaya and TSA for non-rhizobial isolates. The endophytes were then screened for various growth promoting traits like IAA production, Phosphate solubilization, Siderophore production, ACC utilization, Potassium solubilization and tolerance to NaCl concentrations. Total six endophytes, one rhizobium (HM2) and five non-rhizobial endophytes (RE6, BE13, ME3, HE5, HE7) were selected based on of plant growth promoting traits. Furthermore, the most promising non-rhizobial endophytes RE6 and BE13, compatible with Rhizobial isolate HM2 were Pseudomonas protegens and Bacillus boroniphilus using 16s rDNA sequencing. Both the isolates are non-pathogenic to humans and thus, are potential plant probiotics that can be used as biofertilizers.

Desert plants are able to survive under harsh environmental stresses inherent to arid and semiarid regions due to their association with bacterial endophytes. However, the identity, functions, and the factors that influence the association of bacterial endophytes with desert plants are poorly known. These bacterial endophytes can be used as an untapped resource to favor plant growth and development in agro-ecosystems of arid regions. The present study is therefore focused on the isolation and identification of bacterial endophytes from two native medicinal plants (Fagonia mollis Delile and Achillea fragrantissima (Forssk) Sch. Bip.) growing spontaneously in the arid region of the South Sinai (Egypt), and characterization of their plant growth promoting (PGP) traits. Thirteen putative bacterial endophytes were isolated from the leaves of both plant species and characterized for their plant growth promoting abilities using molecular and biochemical approaches, as well as greenhouse trials. Selected endophytic bacterial strains were applied to maize plants (Zea mays L. var. Single cross Pioneer 30K08) to further evaluate their PGP abilities under greenhouse conditions. Isolated bacterial strains have variable plant growth promoting activities. Among these activities, isolated bacterial endophytes have the efficacy of phosphate solubilizing with clear zones ranging from 7.6 ± 0.3 to 9.6 ± 0.3 mm. Additionally, the obtained bacterial endophytes increased the productivity of indole acetic acid (IAA) in broth media from 10 to 60 µg·mL−1 with increasing tryptophan concentration from 1 to 5 mg·mL−1. Bacillus and Brevibacillus strains were frequently isolated from the leaves of both plant species, and had significant positive effects on plant growth and shoot phosphorus (P) and nitrogen (N) contents. Results suggest that these endophytes are good candidates as plant growth promoting inoculants to help reduce chemical input in conventional agricultural practices and increase nutrient uptake and stress resilience in plant species.

Endophytes with plant growth-promoting activity can improve the health and development of plants during all life stages. However, less is known about their stability and transmission across plant genotypes, habitats, and generations. By combining community and isolate analyses, we found that each plant habitat and genotype harbored distinct bacterial communities and plant growth-promoting bacteria (PGPB). Soil, root endosphere, and rhizosphere were the habitats with the highest bacterial diversity, while seeds hosted more selective communities. Seeds generated under field conditions showed traces of a bacterial community composition connected to the suppression of plant pathogens. In contrast, seeds of the successive generation grown in a pathogen-free and low-nutrient environment showed a predominance of bacteria that facilitate the uptake of nutrients. These modifications of the microbiome can be explained by an adaptation to prevalent environmental conditions. Cultivation approaches revealed microhabitat-specific PGPB that were assigned to various species of Bacillus, Stenotrophomonas, and Ralstonia. Tracking down these bacteria among the whole tomato plant allowed us to identify the seed as a primary vehicle of PGPB transmission. This previously undescribed vertical transmission of PGPB represents a strategy to maintain plant beneficial bacteria over generations and has an impact for the design of seed treatments.

Endophytic bacteria form a symbiotic relation with plants and generally cause no harmful effects to the host plants. In a previous study, we isolated eight bacterial endophytes from sweet potato plants harvested in Salyan, Nepal. These endophytes showed plant growth-promoting properties as a mixed culture. In this study, we evaluated the ability of these strains to produce indole-3-acetic acid (IAA) and to fix nitrogen. Based on these results, we selected two strains, Klebsiella sp. Sal 1 and Enterobacter sp. Sal 3, and evaluated their ability to promote plant growth. IAA production activity peaked at 15–60 mg NH4NO3/L in plant-free medium. Similarly, acetylene reduction activity peaked at 0–6.25 mg NH4NO3/L. Both strains successfully colonized plants, promoted the growth of tomatoes, and induced phenotypes in plants consistent with IAA exposure. This suggests that these strains promote plant growth by producing IAA inside the plant, where nitrogen levels are expected to be low.

The application of beneficial bacteria may present an alternative approach to chemical plant protection and fertilization products as they enhance growth and resistance to biotic and abiotic stresses. Plant growth-promoting bacteria are found in the rhizosphere, epiphytically or endophytically (Plant Growth Promoting Endophytic Bacteria, PGPEB). In the present study, 36 out of 119 isolated endophytic bacterial strains from roots, leaves and flowers of the pharmaceutical plant Calendula officinalis were further identified and classified into Bacillus, Pseudomonas, Pantoea, Stenotrophomonas and Rhizobium genera. Selected endophytes were evaluated depending on positive reaction to different plant growth promoting (PGP) traits, motility, survival rate and inhibition of phytopathogenic fungi in vitro and ex vivo (tomato fruit). Bacteria were further assessed for their plant growth effect on Arabidopsis thaliana seedlings and on seed bio-primed tomato plantlets, in vitro. Our results indicated that many bacterial endophytes increased seed germination, promoted plant growth and changed root structure by increasing lateral root density and length and root hair formation. The most promising antagonistic PGPEB strains (Cal.r.29, Cal.l.30, Cal.f.4, Cal.l.11, Cal.f.2.1, Cal.r.19 and Cal.r.11) are indicated as effective biological control agents (BCA) against Botrytis cinerea on detached tomato fruits. Results underlie the utility of beneficial endophytic bacteria for sustainable and efficient crop production and disease control.

Endophytic bacteria are responsible for improved plant growth due to its role in nitrogen fixation, indole acetic acid (IAA) production, phosphate solubilization etc and in plant protection through various mechanisms and production of bioactive compounds. The purpose of this study was to determine the plant growth promoting potential of endophytic bacteria isolated from medicinal plants namely, Adulsa, Amla, Bael, Kadamb, Mango, Neem, Tulsi. Endophytic bacteria isolated from the medicinal plants, comprised of 68% Gram positive and 29% Gram negative bacteria. Seventeen distinctly unique Gram-negative endophytes were selected for further analysis. The selected endophytes were tentatively identified as Pseudomonas sp. The multifarious endophytes were capable of nitrogen fixation, phosphate solubilisation, indole acetic acid (IAA) production, production of antimicrobial compounds and aromatic compound degradation. Some of the endophytic strains were found to harbor plasmids that may play a role in aromatic compound degradation. This study emphasizes the potential of endophytic Pseudomonas species in enhancing plant growth and plant protection.

Echeveria laui (Crassulaceae) is commonly commercialized due to its drought-tolerance capacity and to its rosette-shaped aesthetics. Since endophytes associated with plants from a dry or arid environment have scarcely been analyzed as yet, current research comprises the isolation of leaf endophytic bacteria from E. laui (one five-year-old and one two-year-old plants) investigating plant growth-promoting endophytic bacteria which may solubilize phosphate, fix nitrogen, produce exopolysaccharides/IAA and antagonize phytopathogens. Isolation by the maceration methodology provided a colonization rate of 1.98 x109 CFU g-1 for the two-year-old plant and 1.14 x 1010 CFU g-1 for the five-year-old one. All 40 isolates evaluated showed in vitro plant growth-promoting agent’s abilities, with emphasis on EG04, ELG18, and ELP06. The capacity of the three best bacterial isolates were evaluated under greenhouse conditions in common and black bean (Phaseolus vulgaris L.) plants. Based on the sequencing of the 16S rRNA region and phylogenetic analysis, the three endophytes were identified as Pantoea sp. (ELG04 and ELG18) and Erwinia sp. (ELP06). Under greenhouse conditions, statistically significant differences were found among the plants treated with the three endophytes when compared to control plants for fresh and dry shoot, root biomass and length.

It is a known fact that the whole agriculture system is suffering from the diseases caused by plant pathogens, affecting negatively the crop yield production, food security, biodiversity, agricultural ecosystem and hence agricultural economy. In many countries, the containment strategies of plant pathogens are still depending on chemical pesticides that cause adverse effects in the long term. According to the implementations reinforced by European council 2009/128/EC, biocontrol strategies are considered as the most profound and integrated approach for sustainable disease management. Defining biocontrol in terms of plant pathology, it is the purposeful utilization of beneficial microbes, or its molecules, to suppress phytopathogens’ ability to colonize or induce symptoms in the host. In spite of their lesser shelf-life and unreliability as compared to conventional pesticides, their targeted biological interaction with the phytopathogens reduces the possibility of affecting non-target organisms, environment and the development of resistance in the pathogen. In this context, exploitation of bacterial endophytes has gained much attention during the past decades. Endophytic plant growth promoting bacteria (ePGPBs) mediate their biocontrol efficacy by targeting species through a multitude of direct or indirect biological interactions, often employing both modes of action, such as plant growth promotion, host’s resistance induction, allelochemicals secretion, and nutrients and niche competition. Another strategy that has gained popularity is the exogenous application of double stranded RNA (dsRNA), which is considered as the key trigger molecule of RNA interreference (RNAi), a post-transcriptional gene silencing mechanism, and has been shown to provide protection without the need for integration of dsRNA-expressing constructs as transgenes. In the present doctoral thesis, the above-mentioned biocontrol strategies were adapted, utilizing “ePGPBs as microbial inoculants” and “exogenously applied dsRNA as RNAi based natural product”, against several phytopathogens belonging to different families of viruses and fungi. Regarding ePGBPs as microbial inoculants, the objective of this study was to extend our understanding of five endophytic bacterial strains; Pantoea agglomerans (255-7), Pseudomonas syringae (260-02), Lysinibacillus fusiformis (S4C11), Paraburkholderia fungorum (R8), Paenibacillus pasadenensis (R16); that have shown a promising result in previous studies. In the present doctoral study, these strains were tested in planta to evaluate their role in providing plant growth promotion and broad-spectrum protection against two target pathosystems (viruses and fungi) that might have direct, indirect or simultaneous effects, proceeded with two following aims: (Aim 1) action against viruses: Cymbidium ringspot virus (CymRSV), Cucumber mosaic virus (CMV), Potato virus X (PVX), and Potato virus Y (PVY) on Nicotiana benthamiana plants, comparing their effects with those of three chitosan-based products, which are known to induce resistance in plants; and (Aim 2) action against fungal pathogens: Rhizoctonia solani, Pythium ultimum and Botrytis cinerea on Lactuca sativa plants, comparing their effects with Bacillus amyloliquefaciens strain (CC2) and Trichoderma spp. based product, under controlled conditions. To test the priming efficacy of ePGPBs against target viruses, several phenotypic parameters were observed along with the evaluation of three plant defense related genes (EDS1, PR2B and NPR1) on Nicotiana benthamiana plants. Interestingly, the symptoms reduction was successfully registered against CymRSV and CMV with increased heights of the plants. Some of the treatments were shown correlation between severity of symptoms and the virus concentration in the plants. Furthermore, the molecular interaction indicated the involvement of a salicylic acid (SA) mediated defense pathway as evidenced by the increased expression levels of EDS1 gene in strains R16 and 260-02. Whereas, strain S4C11 showed downregulation of PR2B gene, suggesting that SA-independent pathways could be involved. These findings opened queries regarding the duration of the protective effect, host-plant- pathogen interaction, and epidemiological implications of the use of similar biocontrol strains, that reduce the symptoms but not the concentration of virus in the host. To test the ePGPBs role against target fungi (pre- and post-harvest stage), several experiments were conducted including phenotypic paraments, gene expression analysis (PR1, PAL, ThlP3, ERF1 and ACCS1), microbiota analysis in bulk soil, rhizosphere, and root associated with Lactuca sativa in the presence or absence of the inoculants, and nutritional quality parameters at time of harvest and during shelf-life of Romaine lettuce. The results were accompanied in terms of symptoms reduction by strain R16 (P. ultimum, R. solani, B. cinerea) and strain 260-02 (R. solani, B. cinerea); % seed germination by strains R16, 260-02, 255-7, S4C11 in some healthy and R. solani infected lettuce varieties; inhibition of R. solani population in soil and rhizosphere soil by strains R16, 260-02 and 255-7. Furthermore, composition of the bacterial microbiota was radically different in the rhizosphere and the root endosphere among treatments, while the bulk soil formed a single cluster regardless of treatment, indicating that the use of these treatments did not have an ecological impact outside of the plant. Also, these strains were able to contribute to the maintenance of nutritional quality indexes of lettuce at harvest and during storage. All the obtained results indicated that these strains were involved directly (via antibiosis) and indirectly (via SA or ET/JA) in the observed reduction of symptoms. Particularly, strain R16 upregulated both PAL and ACCS1 gene in R. solani infected L. sativa (suggesting co-activation of SA- and JA/ ET mediated ISR resistance); strain 260-02 upregulated PAL gene in R. solani infected romaine lettuce and showed higher levels of ascorbic acid (AsA) production in B. cinerea infected romaine lettuce (suggesting the activation of SA- and AsA-mediated antioxidant resistance); and strain 255-7 triggered PAL and ThlP3 gene up-stream expression levels indicating SA mediated pathways in R. solani infected romaine lettuce. These findings affirmed the previous conclusions and added valuable pieces of information regarding the traits these ePGPB carried, most importantly, in individuating different mode of action of the different strains in different host plants with or without the presence of pathogen. Regarding the second approach, non-transgenic strategy was employed to induce resistance against Tomato Aspermy Virus (TAV) in N. benthamiana plants. DsRNA molecules for coat protein (CP) gene was produced by a two-step PCR assay followed by in vitro transcription and purification and was exogenously applied. The implementation of CP-derived dsRNA TAV was not successful in reducing observed symptoms (mosaics, blisters, crinkling, leaf distortion, and systemic vein clearing), regardless of treatments or days of post inoculation. Only a slight difference was found in plant heights indicating that the treatment managed to reduce stunted growth of the plant at dilution 01:10 (6 and 12 dpi). The reasons could involve inappropriate concentration of dsRNA inoculum. Therefore, future studies will be conducted to optimize in vitro dsRNA molecules production to obtain higher concentrations or more specific sequences, and more suitable viral genes. Both strategies have shown interesting outcomes and gave us the future direction which will help us in designing the adequate trials (in planta or semi-field) for the disease management and diseases control through the application of ePGPBs as a microbial inoculants and dsRNA-based product individually or in combination.

Magister Scientiae - MSc (Biotechnology)
The yearly increase of global population will result in a greater demand for crop production, but with the climates changes and a lack of available agricultural land it will become increasingly more difficult to provide sufficient crops to feed everyone adequately. Application of the PGPE has proven over the past researches to be able enhance growth of plants via various growth promoting mechanisms. To identify suitable growth promoting bacteria candidate, E. plantagineum plant was used to isolate endophytes from the root after surface sterilization. The isolates bacteria were used to inoculate Brassica napus L seeds. The effects of isolate's ability to promote growth were evaluated based on the certain growth parameters after 42 days in the green house. Isolate CP5 produced highest results in all growth parameter. Isolates CP5 was selected as potential candidate as significant improvement was shown by this isolate. This isolate was tested for the ability to produce ACC deaminase, solubilize phosphate, synthesize IAA and siderophore production. Furthermore isolate CP5 growth promotion abilities was tested on Brassica napus L under antimony stress.
2021-08-31

Magister Scientiae - MSc (Biotechnology)
Endophytic bacteria are known to have an endosymbiotic relationship with plants and provide them with many beneficial properties. These bacteria stimulate plant hormones, provide protection from pathogens and increase nutrient availability in the environment. In this study some of these potential growth factors were tested. Endophytic bacteria have the potential to be of great value for the increase of crop production. They offer a variety of processes that aid in plant growth promotion in an ecofriendly manner. The use of endophytic bacteria provides a cheaper and cleaner approach compared to industrial made fertilizers. They also have potential uses in bioremediation to clean the environment polluted by industrial processes. Endophytes were isolated and showed significant growth improvement. Each isolate displayed different morphologies. Isolates were tested for classical growth promotion mechanisms such as the ability to solubilize phosphate, Indole-3-acetic acid and siderophore production. Inductively Coupled Plasma Optical Emission Spectrometry was performed to measure the effect of the isolates on the plants nutrient profile. The isolates were then tested again while the plants were under heavy metal stress to determine if they were still capable of growth promotion. The plants were then assayed for cell death using Evans blue and biomass was measured to determine the effect of vanadium stress. Inductively Coupled Plasma Optical Emission Spectrometry was performed again to assess the change in nutrient profile while under vanadium stress.
2021-08-31

This thesis reports the results obtained during the three years PhD course focused on the study of culturable bacterial endophytes of Vitis vinifera Glera and their beneficial activities. The study, part of a large project named “EndoFlorVit project” (FEARS-UE and Regione Del Veneto), aims at investigate the biodiversity and the plant growth promoting activities of culturable endophytes isolated from Glera grapevine in vineyards of Conegliano-Valdobbiadene DOCG production area. This thesis reports the results of the isolation of culturable bacterial endophytes from surface-sterilized Glera grapevine tissues. 381 culturable strains were successfully isolated from roots, shoots and leaves of Vitis vinifera Glera, sampled from six different vineyards in the Conegliano-Valdobbiadene DOCG area (Veneto, Italy). The community was investigated by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and nucleotide sequencing to identify the most representative genera of the Glera microbiome. Approximately 30% of the isolates belonged to the genus Bacillus, which was the most represented; other genera such as Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus and Agrococcus were identified. The composition of the communities isolated from different vineyards was not the same; moreover we reported that endophyte biodiversity inside plants was influenced by the season. After molecular characterization, we focused our attention to investigate the plant growth promoting abilities of the culturable strains. Using biochemical tests we assayed some of the most important and effective properties in order to investigate the physiology of these bacteria and identify some strains that could have a strong beneficial effect on plant nutrition and growth. In this work, using Carboxymethyl Cellulose degradation test, we demonstrated that 85 strains secreted cellulolytic enzymes; this trait could confer to these bacteria an advantage in plant penetration and tissues colonization. By qualitative biochemical assays, we demonstrated that many strains were able to solubilize phosphate (127 strains), produce ammonia (142 strains) and secrete siderophores (155 strains). Using the colorimetric Salkowsky assay, we determined that 17 strains produced the phytohormone Indol 3-acetic acid (IAA) ; using Arabidopsis thaliana DR5:GUS, where the β-glucuronidase reporter gene is expressed under control of a IAA-induced promoter, we demonstrated that bacterial IAA was recognised by the Arabidopsis plants and caused morphological alteration on the root architecture. It is known that IAA is not the only bacterial molecule that influence the plant growth and the root morphology. To investigate the effects of the Glera endophytes on the plant morphology we used the model system Arabidopsis thaliana co-cultured in vitro with every single strain. Morphological parameters (root length, surface and diameter) were measured by a software and statistically analysed by cluster analysis. Plants were thus clustered according with the effect of the strain on the root parameters, demonstrating the effects of the strains on roots morphology. In particular, some strains caused an enhanced root length displaying a plant growth promotion effect. By this large-scale characterization we selected two of the most promising strains, one for the putative plant growth effect (Pantoea agglomerans GL83) and one as putative biocontrol agent (Bacillus licheniformis GL174) that were transformed with a DNA cassette containing a gfp reporter gene. Using Laser Scanning Confocal Microscopy, we demonstrate the colonization of the stem endosphere of Glera cuttings 20 and 30 days after the inoculum of the fluorescent strains. In this thesis, the evidences of the colonization are reported demonstrating that Pantoea agglomerans GL83 and Bacillus licheniformis GL174 are true Glera endophytes able to colonize cuttings when re-inoculated. After we demonstrated that B. licheniformis GL174 is a true endophyte of Glera, we investigated the biocontrol abilities of the strain. Results of antagonism tests against plant pathogenic fungi are shown, demonstrating that the strain is able to reduce and inhibit the mycelia growth of the grapevine pathogens Phaeoacremonium aleophilum, Phaeomoniella spp., Botryosphaeria spp., Botrytis cinerea and for the more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. After that, we demonstrated by PCR and DNA sequencing that GL174 has the operons coding for lipopeptide synthetase enzymes and that the strain produced cyclic lipopeptide belonging to surfactin and lichenysin families. These molecules with antimicrobial effects were identified and characterized by mass spectrometric analysis and the results are reported and discussed in this thesis. The genome of the strain was thus sequenced to better investigate the strain and the sequences were preliminary analysed identifying the presence of many genes coding for lytic enzymes. The production of lipopeptides, the inhibition of fungal growth and the ability to colonize inner tissues of Glera indicated GL174 as a good candidate for biocontrol. Another aspect of endophyte-plant symbiosis that is not well explained is the ecology of these bacterial strains and the interactions between different bacterial species in the rhizosphere and inside plants are poorly described. Bacteria-bacteria interactions are likely to be an important factor that defines the composition of the endophyte community. The study of these interactions is essential to understand plant-bacteria relationship; moreover, the study of how the native community of rhizobacteria and endophytes may change after the inoculation of other bacteria is important for a safe and aware use of commercial biocontrol or bio-fertilizer products containing endophytes. A preliminary ecological study is presented in this thesis: some ecological aspects of endophyte of grapevine, endophytes of other plant species and some bacteria commercialized as beneficial strains, called “biofector strains” were analysed using tomato, a model plant for agriculture and horticulture. In this work, we demonstrated that these strains were able to colonize tomato plants and the population densities of the diverse tissues sampled are reported in the Chapter number 5. This work, that is still ongoing, aims to evaluate the impact of these endophytic strains investigating if the inoculum of the bacteria on tomato plants leads to a different endophytic community in comparison to uninoculated plants. This study is essential to unravel the effects of the bio effector strains on natural endophytic populations of plants: from peeled stems of all the inoculated plants the total DNA was extracted; this material will be used as template for the 16S rDNA amplification of all the endophytes present in the plants. Many sets of primers are being tested to select the best combination for this approach. The amplicons will be sequenced and analysed to determine if the community of endophytes has been changed by the inoculum of the external endophytic bio-effector strain. In conclusion, the results presented in this thesis are an overview of the composition of the endophytic community of Glera plants cultivated in the Conegliano-Valdobbiadene DOCG area. The isolation of the culturable strains has provided a large collection of bacteria that, during the PhD course, was characterized investigating plant growth promoting activities and bacteria effects on plant morphology considering different mechanisms underlying plant-microbe interactions. The evidence obtained in this work describes a clear and novel background to understand Glera endophytes biology and ecology and, when confirmed in planta by field trials, will permit the selection of some efficient strains to use as safe endophytic bio-fertilizers and biocontrol agents, for a sustainable production of Glera grapes.
Questo lavoro di tesi presenta e discute i risultati sperimentali ottenuti durante il corso di dottorato in Biologia Evoluzionistica presso la Scuola di Dottorato di Bioscienze e Biotecnologie dell’Università degli Studi di Padova. Questa ricerca, parte di un progetto più ampio denominato “EndoFlorVit” (FEARS-UE e Regione del Veneto), ha come scopo la caratterizzazione molecolare e lo studio delle proprierà di promozione della crescita vegetale e di bio-controllo di batteri endofiti isolati da piante di Vitis vinifera di cultivar Glera, coltivate nell’area di produzione del Prosecco di Conegliano-Valdobbiadene DOCG. Le comunità di microrganismi isolate sono state studiate utilizzando la tecnica ARDRA (Amplified Ribosomal DNA Restriction Analysis) e il sequenziamento di porzioni del 16S rDNA identificando che circa il 30% dei ceppi isolati appartengono al genere Bacillus, il quale risulta essere il più rappresentato nelle piante campionate. Altri generi a cui appartengono numerosi ceppi isolati sono Staphylococcus, Microbacterium, Paenibacillus, Curtobacterium, Stenotrophomonas, Variovorax, Micrococcus e Agrococcus. La composizione delle comunità endofite isolate da differenti piante non è uniforme: esse variano nei differenti vigneti e sono inoltre influenzate dalla stagionalità. Oltre alla descrizione dei ceppi isolati, in questo lavoro di tesi sono presentati e discussi i risultati dello studio delle proprietà di promozione della crescita vegetale dei ceppi isolati. Utilizzando saggi biochimici sono state investigate alcune delle principali attività benefiche che hanno un effetto di miglioramento della nutrizione vegetale. Mediante il test di degradazione della carbossimetil-cellulosa sono stati identificati 85 ceppi capaci di secernere enzimi degradanti la cellulosa: questa capacità può conferire ai ceppi che la esprimono un vantaggio nella colonizzazione dei tessuti vegetali facilitando loro il processo di penetrazione nei tessuti della pianta. Attraverso saggi biochimici qualitativi è stato possibile dimostrare che numerosi ceppi sono in grado di solubilizzare il fosfato insolubile (127 ceppi), produrre ammoniaca (142 ceppi) e secernere siderofori (155 ceppi). Inoltre, utilizzando il saggio di Salkowski, è stato dimostrato che 17 ceppi batterici producono l’ormone vegetale Acido 3-indolacetico (IAA). Per investigare l’effetto dei ceppi produttori di IAA sulla fisiologia e morfologia della pianta è stato utilizzata la pianta modello Arabidopsis thaliana DR5:GUS, una linea mutante esprimenti l’enzima β-glucuronidasi sotto controllo di un promotore indotto da IAA. Utilizzando questo sistema sperimentale è stato dimostrato che l’IAA prodotto dai batteri viene riconosciuto dalle piante di Arabidopsis e causa alterazioni alla morfologia e architettura radicale. È noto tuttavia che l’IAA non è l’unica molecola batterica che influenza la crescita vegetale e la morfologia vegetale. In tal senso, è stato valutato l’effetto di ciascun ceppo isolato sulla pianta Arabidopsis thaliana Col-0 wild tipe. Tre parametri morfologici della radice (lunghezza superficie e diametro) sono considerati e analizzati statisticamente mediante cluster analysis. Le piante quindi sono state raggruppate secondo gli effetti che i batteri hanno provocato sull’apparato radicale assegnando in questo modo a ciascun ceppo l’effetto corrispondente. In questo modo è stato dimostrato che alcuni ceppi hanno causato allungamento della radice, un effetto ascrivibile come promozione della crescita. Da questa caratterizzazione ed analisi su larga scala dei ceppi isolati sono stati selezionati due ceppi particolarmente promettenti per la promozione della crescita vegetale e per il biocontrollo: Pantoea agglomerans GL83 e Bacillus licheniformis GL174. Questi due ceppi sono stati trasformati geneticamente con un costrutto contenente il gene che esprime la proteina fluorescente GFP. Utilizzando tecniche di microscopia confocale è stato dimostrato che entrambi i ceppi fluorescenti sono in grado di ricolonizzare talee di vite Glera quando inoculate e che persistono all’interno dei tessuti del fusto dopo 20 e 30 giorni dopo l’inoculo. In questa tesi quindi sono presentate le evidenze sperimentali che questi due ceppi, Pantoea agglomerans GL83 e Bacillus licheniformis GL174, sono veri endofiti di vite Glera e risultano quindi interessanti per le loro proprietà benefiche. Dopo aver confermato che GL174 è endofita della vite Glera, il ceppo è stato investigato per evidenziarne alcune capacità utili per il biocontrollo dei patogeni. In questo lavoro di tesi sono presentati i risultati di saggi di antagonismo in vitro nei quali il ceppo in esame ha effetto di inibizione della crescita del micelio di alcuni funghi patogeni della vite (Phaeoacremonium aleophilum, Paeomoniella spp., Botryosphaeria spp., Botrytis cinerea) e di due patogeni più generalisti (Sclerotinia sclerotiorum e Phytophtora infestans). Lo studio del ceppo GL174 si è successivamente focalizzato sulla capacità del batterio di produrre i lipopeptidi ciclici, una classe di molecole con forte attività antimicrobica e surfattante. Per prima cosa, attraverso PCR e sequenziamento del DNA, è stata identificata la presenza nel genoma batterico degli operoni codificanti per alcune lipopepide sintetasi, gli enzimi deputati alla sintesi di queste molecole. La produzione di lipopeptidi è stata successivamente dimostrata utilizzando tecniche di spettrometria di massa; le quali hanno permesso di identificare le molecole prodotte e di ricostruirne la struttura chimica. La produzione di queste molecole e la capacità inibitoria di funghi patogeni rendono il ceppo GL174 un buon candidato come agente di biocontrollo nella coltivazione della vite e di altre specie economicamente rilevanti. L’ ecologia dei batteri endofiti è un tema che ancora non è stato del tutto investigato all’interno dello studio dell’interazione tra piante ed endofiti. Inoltre, le interazioni che avvengono a livello di rizosfera ed endosfera non sono ancora ben descritte ma evidenze sperimentali suggeriscono che esse siano un fattore importante nella definizione della composizione delle comunità endofite. Lo studio di come un inoculo batterico esogeno può modificare la composizione della comunità nativa di endofiti è essenziale per un uso consapevole di formulati commerciali a base di batteri endofiti. In questa tesi viene quindi presentato un lavoro preliminare che analizza l’ecologia di ceppi isolati da Glera, ceppi isolati da altre specie vegetali, e batteri commercializzati come biostimolanti. La colonizzazione di piante di pomodoro, pianta modello per lo studio delle specie orticole, da parte di questi ceppi microbici è stata dimostrata e quantificata per radici, fusto e foglie di piante coltivate per 3 e 5 settimane. Questo lavoro ha come scopo inoltre l’analisi delle comunità di endofiti di queste piante inoculate per confrontarne la composizione con piante non inoculate. Lo studio, che è ancora in corso, ha comportato l’estrazione del DNA totale della endosfera di porzioni di fusto; da questo DNA saranno amplificati i 16S rDNA dei batteri endofiti presenti e sequenziati con tecniche di NGS. In conclusione, i risultati presentati in questa tesi descrivono la composizione delle comunità di endofiti coltivabili isolate da piante di vite Glera della zona del Prosecco Conegliano-Valdobbiadene DOCG. L’isolamento di tali batteri ha fornito una larga collezione di ceppi batterici, le cui proprietà benefiche che promuovono la crescita vegetale e gli effetti dei batteri sulla morfologia radicale di Arabidopsis thaliana sono stati analizzati e presentati criticamente coinvolgendo più aspetti importanti nell’interazione pianta-endofiti. I risultati ottenuti in questo lavoro descrivono le proprietà di alcuni ceppi isolati da Glera che, quando confermato da prove sperimentali in campo, potranno essere utilizzati in sicurezza come agenti endofiti di biofertilizzazione e/o biocontrollo nella produzione dell’uva Glera e di altre specie vegetali economicamente importanti.

Plant growth-promoting bacteria (PGPB) have been studied in many agriculturally interesting plants, but never in pitcher plants. Sarracenia oreophila (the green pitcher plant) is an endangered species in Georgia, Alabama, and North Carolina (Rice 2010). With the help of Dr. Jim Spain's lab, a previous student in Dr. Gerald Pullman's lab discovered evidence that nitrogen-fixing bacteria (Burkholderia spp.) live within these pitcher plants. This study aims to determine whether these nitrogen-fixing bacteria confer a benefit to their host plants by providing fixed nitrogen. To do this, pitcher plants were inoculated with the Burkholderia and grown on a control medium, a medium without sugar (as the sugar causes the bacteria to grow until they hinder the plants), various media that are missing nitrogen-containing compounds usually provided in growth media, and a medium completely lacking nitrogen. These plants were compared to control plants on the same media that had not been inoculated with Burkholderia. The plants' biomass and root growth were measured. The data suggest that Burkholderia may stimulate plant biomass growth when sufficient nitrogen is present and there may be a nitrogen-threshold that needs to be met in order to sustain the Burkholderia-Sarracenia symbiosis. Also, the Burkholderia has a negative effect on roots grown in high-nitrogen media, possibly due to competition for nutrients.

One of the major challenges for agricultural research in the 21st century is to increase crop productivity to meet the growing demand for food and feed. Biotic (e.g. plant pathogens) and abiotic stresses (e.g. soil salinity) have detrimental effects on agricultural productivity, with yield losses being as high as 60% for major crops such as barley, corn, potatoes, sorghum, soybean and wheat, especially in semi-arid regions such as Saudi Arabia. Plant growth promoting bacteria isolated from pioneer desert plants could serve as an eco-friendly, sustainable solution for improving plant growth, stress tolerance and health. In this dissertation, culture-independent amplicon sequencing of bacterial communities revealed how native desert plants influence their surrounding bacterial communities in a phylogeny-dependent manner. By culture-dependent isolation of the plant endosphere compartments and a number of bioassays, more than a hundred bacterial isolates with various biochemical properties, such as nutrient acquisition, hormone production and growth under stress conditions were obtained. From this collection, five phylogenetically diverse bacterial strains were able to promote the growth of the model plant Arabidopsis thaliana under salinity stress conditions in a common mechanism of inducing transcriptional changes of tissue-specific ion transporters and lowering Na+/K+ ratios in the shoots. By combining a number of in vitro bioassays, plant phenotyping and volatile-mediated inhibition assays with next-generation sequencing technology, gas chromatography–mass spectrometry and bioinformatics tools, a candidate strain was presented as a multi-stress tolerance promoting bacterium with potential use in agriculture. Since recent research showed the importance of microbial partners for enhancing the growth and health of plants, a review of the different factors influencing plant-associated microbial communities is presented and a framework for the successful application of microbial inoculants in agriculture is proposed. The presented work demonstrates a holistic approach for tackling agricultural challenges using microbial inoculants from desert plants by combining culturomics, phenomics, genomics and transcriptomics. Microbial inoculants are promising tools for studying abiotic stress tolerance mechanisms in plants, and they provide an eco-friendly solution for increasing crop yield in arid and semi-arid regions, especially in light of a dramatically growing human population and detrimental effects of global warming and climate change.

The endophytic genus plant growth promoting bacteria (EPGPB) known as Xanthobacter autrotrophicus is one of the most interesting option to apply on the production of wheat (Triticum aestivum), and other domestic crops lettuce (Lactuca sativa), tomato (Solanum lycopersicum) rice (Oriza sativa) maize (Zea mays): under all types of agriculture systems: open field, protecting one or either organic sustainable type. The aims of this review is to analyze the qualities of X. autotrophicus as useful EPGPB for sustainable production of wheat and other crops regarding its capacity as able to fix molecular nitrogen (N2) as well as by transforming plant metabolic compounds in phytohormons, including phosphatase enzyme for solubilizing phosphate to solve different soil problems related with its fertility also some phytopathological like to stop of growing weed as Arabidopsis thaliana which are competing with health growth of domestic plants. Beside the potencial of X. autotrophicus for bioremediation of environmental polluted by chemicals.

In order to grow, reproduce, and defend themselves, maize plants use various strategies to obtain adaptive advantages in varying conditions, for example, to tolerate abiotic stress (e.g., drought or heat due to climate change). One of these strategies is the establishment of interactions with plant-growth-promoting bacteria. Bacteria can be associated with plants in the rhizosphere, rhizoplane, or as endophytes. Recent evidence suggest that modern agricultural practices are detrimental to these beneficial plant-microbe interactions, and reservoirs like traditional agroecosystems called milpas, emerge as sources of microbiota associated with maize crops, with increased diversity and beneficial functions. Particularly, bacterial endophytes associated with native maize from milpas show promising features for their use as plant-growth-promoting inoculates, however, it is necessary to first understand the mechanisms known for beneficial functions of endophytes associated with maize and other plants. Here, we review the mechanisms of beneficial interactions between plants and endophytic bacteria, with emphasis on maize and with mentions of recent findings on maize landraces from milpa systems.

The exploration of microbial resources is necessary for plant growth promotion, biological control, and reducing the agrochemicals and fertilizers for sustainable agriculture. Bacteria and fungi are distributed in the biosphere including the rhizosphere and help the host plants by alleviating biotic and abiotic stress through different mechanisms and can be used as bioinoculants for biocontrol and plant growth promotion. Actinobacteria are among the most abundant groups of soil microorganisms. They have been studied for their function in the biological control of plant pathogens, interactions with plants, and plant growth promotion. Streptomyces is the largest genus of actinobacteria. Streptomyces acts as both plant growth promoter and also as plant disease suppressor by various mechanisms like an increase in the supply of nutrients such as phosphorus, iron, production of IAA, and siderophore production. Endophytic actinobacteria help in plant growth-promoting through multiple ways by producing plant hormones; controlling fungal disease through antibiosis and competition. This review briefly summarizes the effects of actinobacteria on biocontrol, plant growth promotion, and association with plants as endophytes.