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Endophytes and recovery from grapevine yellows disease
No full textPlants are naturally associated with different kind of microorganisms. Endophytic bacteria or fungi colonize the host plant systemically, without damaging the host or eliciting symptoms of plant disease according to widely used definition (Quispel, 1992). Endophytes have beneficial effects on plants and they may confer plant protection against pathogens by induction of plant defense mechanisms (ISR), pathogens-antagonistic substances and competition for root colonization (Compant et al., 2010). However, the molecular basis of endophytic interactions is not well understood. Even it is not clear the mechanism, when ISR is activated, it is manifested as a reduction in the rate of disease development, resulting in fewer diseased plants or lesser disease severity (van Loon et al 2007). Flavescence dorée (FD) and Bois noir (BN), the two main phytoplasma associated diseases of grapevine yellows complex (GYs), have been seriously damaging the wine production worldwide. In the last decade, in Italian vineyards was observed the spontaneous remission of symptoms in grapevine plants infected by phytoplasmas (Recovery). Although the bases of this phenomenon are still unclear, different hypothesis have been investigated (Musetti et al., 2007; Bulgari et al., 2011; Grisan et al., 2011). One of this assumptions is the role of endophytic bacteria in recovery. With this purpose the endophytic bacterial community associated grapevine plants was characterized by cultivation-dependent and –independent methods. Composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. Length heterogeneity-polymerase chain reaction (LH-PCR) of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure are different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed which LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR electrophoretic peaks, assigned to isolated cultivable single bacterial strains, were used to monitor their distribution in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Interestingly, some isolated strains showed beneficial traits related to mineral nutrition (phosphate solubilization, siderophore production), development (indole acitic acid production) and health (chitinase).
In conclusion, from the above data it can be speculated that the alterations induced by phytoplasmas in the grapevine endophytic bacterial community by selecting those bacterial strains more resistant to ROS and able to eliciting plant defense responses, including ROS as well, may ultimately lead to recovery. This view is supported by previously reported findings showing that recovered grapevine plants have higher level of ROS in respect to diseased and healthy ones (Musetti et al., 2007). In order to verify this hypothesis, future studies will focus on determining the relative abundance of putative recovery inducers within microbial community living in grapevines. Furthermore, the possibility that endophytic bacteria are involved in the recovery phenomenon opens new perspectives in the control of these detrimental diseases and could be a starting point for developing environmental friendly, biocontrol strategies to be used in open field
Endophytic bacteria living in grapevine plants under different phytosanitary conditions
No full textGrapevine yellows (GY), a disease complex associated with phytoplasmas, induce severe crop losses and depreciate the wine quality, therefore their containment is a priority in the European wine producing areas. Up to now, none Vitis vinifera L. varieties have been found resistant to phytoplasma infection. In recent years, there has been an increasing interest about the recovery from GY diseases and in the role of endophytic bacteria as biocontrol agents. Endophytic bacteria can be termed all the bacteria colonizing the interior of plants without inducing diseases, including those that become pathogenic under certain conditions. Though their relationship with the host is not well understood, they may reduce the disease severity by activating systemic resistance, production of allelochemicals (biocidal volatiles, antibiotics, and lytic enzymes) and competition of nutrients and niches. Endophytic bacterial community associated with grapevine leaf tissues was characterized by cultivation-independent analyses (16S rRNA gene library and Length Heterogeneity-PCR) and cultivation methods. In order to identify endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Library analysis of 16S rDNA showed best sequence matches with Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. More than 85% of the cloned sequences yielded best matches with the species Pantoea agglomerans. Bacteria isolation through cultivation showed best sequence matches with Curtobacterium, Stenotrophomonas, Methylobacterium, Pectobacterium, Enterobacter, Brevundimonas, Agrobacterium, Brevibacillus, Staphylococcus, Sphingomonas, Acaricomes, and Enterococcus. Specific electrophoretic peaks, associated with bacterial species identified in this study, were inserted in a reference LH-PCR database. The creation of this database was the first essential step for an extended large survey on bacterial diversity in vineyards in Italy. Preliminary data registered several additional peaks in healthy, phytoplasma-infected and recovered grapevine plants from Lombardy vineyards. Although bacteria identified in the present study probably do not represent the whole microbial diversity in grapevine plants, cultivation-independent approach could be used in order to compare endophytic bacterial communities in different ecological niches and to identify bacteria potential biocontrol agents of GY
PHYTOPLASMAS-ENDOPHYTES INTERACTIONS: THE CASE STUDY OF GRAPEVINE YELLOWS RECOVERY
No full textThis PhD project aims to study the interactions between endophytic bacteria and phytoplasmas associated with grapevines to clarify the role of endophytic bacteria in grapevine yellows (GY) recovery and discuss the possibility to apply these bacteria for GY management. Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. To identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Library analysis of a PCR-amplified 16S rDNA fragment showed best sequence matches with Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Nucleotide sequences of 16S rDNA from bacteria isolated through cultivation showed best sequence matches with Curtobacterium, Stenotrophomonas, Methylobacterium, Pectobacterium, Enterobacter, Brevundimonas, Agrobacterium, Brevibacillus, Staphylococcus, Sphingomonas, Acaricomes, and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Furthermore, composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. LH-PCR of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure is different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed that three LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR database were used to monitor the distribution of bacterial endophytes in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Observed low bacterial richness and different microbial composition in infected and recovered plants suggest that phytoplasma infection could directly and/or indirectly restructure bacterial community selecting endophytic strains that are able to elicit plant defense response. Moreover, we investigated the influence of Flavescence dorée phytoplasmas (FDp) on endophytic bacterial community by studying the seasonal fluctuation of bacterial species associated with healthy, FDp-diseased and recovered plants during phytoplasma infection process. Statistical analysis indicated that, before phytoplasma titre inside diseased plant tissues becomes detectable, endophytic bacterial community is similar to that associated with healthy plants and differs from that associated with recovered plants. Moreover, it seems that a change in microbial composition could be determined when phytoplasmas start to replicate. LH-PCR database showed a seasonal fluctuation of some bacteria identified in the analyzed grapevines; these fluctuations are also related to the presence/absence of the pathogen. On the basis of these evidences we hypothesized that phytoplasma replication determines a change in microbial composition selecting few endophytes that could induce recovery mediated by priming response. Finally, in order to study in which way phytoplasmas interact with endophytes and host plant we developed and tested two different microscopy techniques. Fluorescence in situ hybridisation (FISH) allowed to localise, as expected, phytoplasmas in Catharanthus roseus L. (G. Don) phloem tissues, and also to visualize endophytes in phloem, xylem and parenchyma in the same leaf tissues. On the other hand, immune-confocal laser scanning microscopy was applied for demonstrating that SAP11 (a phytoplasma effector protein) can be unloaded from the phloem by itself. These techniques will be used in combination for clarifying if phytopasmas co-localise with ISR bacterial-inducers and for studying proteins involved in phytoplasmas-endophytes-host cross-talk
Microscopic localization of grapevine phytoplasmas: an exciting challenge or a losing battle?
No full textPhytoplasma localization in grapevine by microscopic techniques has always been a big challenge for many reasons, but particularly for their very low concentration in this plant species that makes their finding an almost impossible task, at least by transmission electron microscopy (TEM) (Faoro, 2005). A literature survey, since the discovery of phytoplasmas associated with Flavescence doreè (FD) and other grapevine yellows (GY) in the sixties, shows that only three papers have been published up to now on this subject (Granata et al., 1991; Meignoz et al., 1992; Credi, 1994), in spite of the huge number of reports dealing with the presence of these prokaryotes in infected grapevine plants and detected by PCR techniques. Indeed, by the advent of molecular biology in the eighties, microscopic visualization of phytoplasmas in infected plants was regarded as a useless diagnostic tool, in any case providing very little information on these microorganisms, particularly from the taxonomic point of view. For all the above reasons microscopic investigations on grapevine yellows were completely abandoned. However, in the last decade the numerous efforts that have been carrying out to study the interaction of phytoplasmas with grapevine tissues, particularly in case of the recovery phenomenon, have shown that the precise localization of the these prokaryotes in the tissues would be determinant to understand the underlying mechanisms. Recovery, i.e. the spontaneous remission of symptoms in diseased plants, has often been observed in FD- and GY-affected grapevines (Caudwell et al.,1961; Osler et al., 1999). This phenomenon may or may not involve the elimination of the pathogen from the host. Physiological mechanisms and possible biological factors involved in recovery are still not clear, though increased hydrogen peroxide level in the phloem of recovered plants has been observed (Musetti et al., 2007), together with the activation of systemic acquire resistance related genes (SAR) (Albertazzi et al., 2009). Moreover, other researchers hypothesized that endophytic microorganisms (bacteria, fungi, and mycorrhiza) associated with plant tissues can take a part in the recovery phenomenon (Romanazzi et al., 2009, Bulgari et al., 2011b).
While studying the role of endophytic bacteria in inducing recovery we have faced the need of verifying the distribution of both phytoplasmas and bacteria in grapevine tissues to shed light on their interaction and, in particular, to exclude their direct competition in the phloem cells (Bulgari et al., 2011a). For this reason we resumed microscopic techniques, such as TEM, coupled with fluorescent in situ hybridization (FISH), a method we previously successfully applied to co-localize grapevine phytoplasmas and endophytic bacteria in the host plants Catharanthus roseus (Bulgari et al., 2011a).
Materials and Methods
Leaf midribs from healthy, FD-diseased and recovered grapevine plants (cv. Cabernet Sauvignon) were collected in summer 2010 and 2011 and processed for conventional TEM analysis and FISH, as previously described (Faoro et al 1991; Bulgari et al. 2011a). Portion of the samples were also analyzed by PCR to confirm the presence of 16SrV phytoplasmas and/or endophytic bacteria. To localize phytoplasmas with FISH, a probe targeting 16SrV group, labeled with FAM (Primm, Milan Italy) or Marina Blue (MB) (Invitrogen, Milan Italy) at 5’ terminus was used. Both these dyes, emitting respectively at 518 nm and 459 nm were tested, to find out the appropriate wavelengths that interfere at least with leaf auto-fluorescence. Endophytic bacteria localization was performed with a universal probe targeting bacterial 16S rDNA (but not phytoplasmal DNA), labeled with a fluorophore (Cy-5) emitting in the far-red (670 nm) (Bulgari et. al 2011a). Labeled sections were observed with a videoconfocal microscope (Nikon, Vico, Italy).
Results and Discussion
Tem analysis, in spite of the nowadays ameliorated procedure in specimen preparation, confirmed that is not a suitable technique for phytoplasma visualization in grapevine tissues. In fact, phytoplasma detection in thin sections was an almost extraordinary event, also in heavily infected grapevine plants (Fig. 1), in spite of the severe ultrastructural alterations present in the phloem tissue. This is possibly due to the ease with which phytoplasma disruption occurs in grapevine deranged phloem. Even endophytic bacteria localization was not easy by TEM, because these prokaryotes were scattered throughout different tissues and not grouped in enclaves. FISH technique encountered some problems in phytoplasma end endophytic bacteria visualization, mainly due to phloem autofluorescence which was particularly heavy in infected plants (Fig. 2), because of polyphenols deposition. These compounds emitted in both the wavelengths of FAM and MB, thus they were difficult to differentiate from the probes. Only Cy5 was shown to be an excellent reporter molecule for in situ hybridization analysis in grapevine tissues, as it emission was in a band far away from that of polyphenols. With the Cy5 probe it was possible to localize endophytic bacteria in all the examined samples (healthy, diseased and recovered), mostly in the xylem but also in the phloem tissues as scattered spots (Fig. 3), suggesting their random distribution, without large accumulation in specific cells. Instead, phytoplasma probes gave less clear cut results and only in a few cases it was possible to observe specific fluorescence of the probe in the phloem of diseased plants (Fig. 2), but not in healthy or recovered ones. From these results, although too preliminary to drawn any suggestion on phytoplasma-endophytic bacteria interaction in grapevine tissues, it can be concluded that FISH remains the only microscopic technique that have some chance in localizing the very few phytoplasmas present in diseased plants, thus allowing to study their interaction with endophytic bacteria. However, further investigations are needed to improve the technique, particularly for the reduction of tissue autofluorescence that would permit to label probes with a larger set of fluorophores and to co-localize phytoplasma and endophytic bacteria together in the same section
Streptomycetes against Colletotrichum circinans: a new way to control anthracnose in onion?
Full text linkColletotrichum circinans is an important plant pathogenic fungus that causes anthracnose, or “smudge”, in onion. Bulbs for pickle production have very high standards for the aesthetic quality of the products. Anthracnose causes symptoms that are not compatible with industrial processing, leading to significant losses to producers. The number of available chemical molecules for its control is limited, and hardly would give acceptable results in the control of soil-borne diseases. Additionally, their use is restricted by the need to consume the product at early stages of growth reducing the interval from treatment to consumption.
The use of alternative and environmentally friendly soil treatments is therefore necessary to control the disease. A set of Streptomyces spp. strains (n= 30) was tested for their activity against four strains of Colletotrichum circinans. In vitro antibiosis assays were coupled with in vivo greenhouse and field trials with natural disease occurrence. Overall, four strains showed promising growth-inhibitory activity against C. circinans, reducing its growth by up to 56%. In in vivo controlled conditions, streptomycetes applied in the soil showed the ability to reduce the incidence of the disease by more than 70%. In field trials, the disease incidence was reduced by up to 57%, and disease severity decreased from 31.33% to 12.33% when using the DEF48, DEF39, and DEF58 strains compared to the untreated control. Influence on plant growth was also observed on onion plants treated especially with Streptomyces spp. DEF19 and DEF58.
Overall, the use of streptomycetes to control soil-borne diseases is promising. Optimization of the delivery methods for the strains may further improve their efficacy
Characterization of beneficial bacteria isolated from grapevine leaves
No full textBacterial endophytes are plant-associated bacteria that affect the plant life cycles in different manners such as nitrogen fixation or the biocontrol of plant pathogens (Lugtemberg and Kamilova 2009). A basic point for the success of sustainable management of plant diseases based on biocontrol agents is the study of endophytic bacterial community associated with plants. Recently endophytic bacteria associated with healthy, grapevine yellows (GY)-diseased and recovered grapevine plants have been described suggesting some putative biocontrol agents (Bulgari et al., 2009; 2011). In the present work, endophytic bacteria isolated from healthy diseased and recovered grapevine plants were characterized for five beneficial traits related to mineral nutrition (phosphate solubilization, siderophores), development (indolacetic acid-IAA synthesis), stress relief (1-amino-cyclopropane-1-carboxylate deaminase and catalase activity), disease control (chitinase activity, siderophores). In detail, five bacterial isolates showed the ability to solubilize phosphate, react to stress and to synthesize IAA. In addition, some of these strains are able to produce siderophores. Furthermore, the distribution of three bacterial genera was analyzed in healthy, GY-diseased and recovered grapevine plants sampled from June to October. At the genus level we found a different bacterial distribution related to the season and to sanitary status.
In conclusion, we have characterized and detected beneficial bacterial strains from grapevine leaves which have the potential to enhance plant growth and suppress diseases
Detection of phytoplasmas and bacterial endophytes in the plant model Catharanthus roseus by fluorescence in situ hybridization.
No full textFluorescence in situ hybridization as a tool for studying phytoplasma-endophytes interaction in plant
No full textBacteria residing in plant tissues without inducing symptoms of diseases are defined as endophytes. They can protect plants from pathogens through three different mechanisms: competition; production of allelochemic inhibitors and induction of systemic resistance (ISR). Recently statistical analysis, carried out on LH-PCR (Length Heterogeneity-PCR) profiles, highlighted a different composition of endophytic bacterial community associated with healthy, GY diseased, and recovered grapevine plants.
In this study we experimented fluorescence in situ hybridization (FISH) to study the interaction between phytoplasmas and endophytic bacteria in the model plant Catharanthus roseus L.. Both phytoplasmas and bacteria were co-localized by confocal microscopy using different probes to target 16SrV phytoplasmal and bacterial 16S rDNA, respectively. To avoid interference with autofluorescence of tissue constituents, bacterial probes were labelled with fluorophores emitting in the far-red (i.e. CY5). Phytoplasmal probes were labelled with FAM and they prove to be able in discriminating genetically different phytoplasmas, localized only in the phloem tissues. Endophytic bacteria were instead detected in the phloem, xylem and leaf parenchyma. These results, though preliminaries, show the great potentiality of FISH in studying the interaction between phytoplasmas and endophytic bacteria
Endophytic bacterial community is restructured in grapevine yellows-diseased and recovered vitis vinifera L. plants : outcome of plant response to phytoplasma infection and starting point for recovery?
No full textFlavescence doree (FD) and Bois noir (BN), grapevine yellows (GY) diseases associated with phytoplasmas, induce severe crop losses. Until now, none grapevine varieties have been found resistant to phytoplasmas infection (Laimer et al., 2009). FD control is based on chemical treatments against the insect vector, Scaphoideus titanus Ball; on the other hand, this strategy is not efficient for BN containment since biological complexity of this disease. In recent years, there has been an increasing interest about the recovery from GY diseases and in the role of endophytic bacteria as biocontrol agents (Bulgari et al., 2009, 2011). Composition and structure of endophytic bacterial community were examined in healthy, phytoplasma-diseased and recovered grapevine plants. Length heterogeneity-polymerase chain reaction (LH-PCR) of total DNA from grapevine leaves was used to generate amplicon profiles that were analyzed with univariate and multivariate statistical methods. Jaccard analyses highlighted that microbial diversity and structure are different in healthy, diseased and recovered grapevine plants. Multivariate analyses confirmed this trend and showed which LH-PCR peaks determined the variation in microbial composition. Furthermore, LH-PCR electrophoretic peaks, assigned to isolated cultivable single bacterial strains, were used to monitor their distribution in total DNAs from analyzed plants. Bacterial community associated with healthy plants was characterized by a greater richness (higher number of LH-PCR peaks) than that present in diseased and recovered plants. Observed low bacterial richness and different microbial composition in diseased and recovered plants suggest that phytoplasma infection can directly and/or indirectly restructure bacterial community selecting endophytic strains that are able to elicit plant defense response
Preliminary results on endophytic bacterial community fluctuation during phytoplasma infection
No full textIn this study we investigated the influence of 'flavescence dorée' 16SrV-C/-D phytoplasmas on endophytic bacterial community by studying the seasonal fluctuation of bacterial species associated with healthy, 'flavescence dorée'-diseased and recovered plants during phytoplasma infection process. Preliminary results showed that, before phytoplasma titre inside diseased plant tissues becomes detectable, endophytic bacterial community is similar to that associated with healthy plants and differs from that associated with recovered plants
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