Endophytic and rhizospheric bacteria associated with Paspalum atratum and its potential for plant growth promotion with different phosphate sources.

The genus Paspalum belongs to the family Poaceae and has several species that are native to Brazil. The Paspalum Germplasm Bank (GB) of the Brazilian Agricultural Research Corporation comprises approximately 450 accessions from 50 species. Among these accessions, Paspalum atratum (BGP 308) has economic potential for forage purposes. However, the endophytic and rhizospheric microbial communities within this accession and their ability to promote plant growth remain unknown. The present study aimed to isolate the endophytic and rhizospheric bacteria associated with P. atratum and to assess their potential for plant growth improvement, so-called plant growth-promoting bacteria (PGPB). For the in vitro tests, the ability of nitrogen-fixing bacteria (NFB), phosphate solubilization (PS) and indoleacetic acid (IAA) production were evaluated. A total of 116 endophytic and rhizosphere bacteria were obtained from the isolation. In the in vitro tests, 43 (37.00%) of these isolates showed positive NFB, PS, and IAA results. These isolates were identified by 16S rDNA sequencing. The phosphate solubilization index (PSI) ranged from 2 to 3.61, all 43 strains performed biological nitrogen fixation and the IAA production ranged from 12.85 to 431.41 µg ml-1. Eight of these 43 isolates were evaluated in vivo in a greenhouse using P. atratum caryopsis. The pots were filled with soil prepared with three different phosphate sources and one control without phosphate. After growth, the plants were submitted to morphological, bromatological and chemical determination. Data were analyzed using analysis of variance (ANOVA) and principal component analysis (PCA). In the in vivo test, treatments 105 (Pseudomonas sp.) and 458 (Pseudomonas sp.) were the most significant for the crystalline phosphate source, 109 (Bacillus sp.) for the sedimentary phosphate source and, as for the soluble phosphate source most treatments that received bacterial isolates had higher phosphorus content in the dry matter than the uninoculated soluble phosphate control. The 105FCR (crystalline phosphate + Pseudomonas sp.), 109FSE (sedimentary phosphate + Bacillus sp.), and 110 FSE (sedimentary phosphate + Enterobacter sp.) treatments showed the best results for plant growth promotion. This work made it possible to determine the bacterial community associated with P. atratum (BGP308) and to obtain new potential plant growth-promoting strains.

The potential of nanomaterials associated with plant growth-promoting bacteria in agriculture.

The impacts of chemical fertilizers and pesticides have raised public concerns regarding the sustainability and security of food supplies, prompting the investigation of alternative methods that have combinations of both agricultural and environmental benefits, such as the use of biofertilizers involving microbes. These types of microbial inoculants are living microorganisms that colonize the soil or plant tissues when applied to the soil, seeds, or plant surfaces, facilitating plant nutrient acquisition. They can enhance plant growth by transforming nutrients into a form assimilable by plants and by acting as biological control agents, known as plant growth-promoting bacteria. The potential use of bacteria as biofertilizers in agriculture constitutes an economical and eco-friendly way to reduce the use of chemical fertilizers and pesticides. In this context, nanotechnology has emerged as a new source of quality enrichment for the agricultural sector. The use of nanoparticles can be an effective method to meet the challenges regarding the effectiveness of biofertilizers in natural environments. Given the novel sustainable strategies applied in agricultural systems, this review addresses the effects of nanoparticles on beneficial plant bacteria for promoting plant growth.

Discovery of a Novel Lineage Burkholderia cepacia ST 1870 Endophytically Isolated from Medicinal Polygala paniculata Which Shows Potent In Vitro Antileishmanial and Antimicrobial Effects.

In this study, we report the isolation and identification of an endophytic strain of Burkholderia cepacia (COPS strain) associated with Polygala paniculata roots. Polygala plants are rich sources of promising microbiomes, of which the literature reports several pharmacological effects, such as trypanocidal, antinociceptive, anesthetic, anxiolytics, and anticonvulsant activities. B. cepacia COPS belongs to a new sequence type (ST 1870) and harbors a genome estimated in 8.3 Mbp which exhibits the aminoglycosides and beta-lactams resistance genes aph(3')-IIa and bla TEM-116, respectively. Analysis performed using MLST, average nucleotide identity, and digital DNA-DNA hybridization support its species-level identification and reveals its novel housekeeping genes alleles gyrB, lepA, and phaC. The root endophyte B. cepacia COPS drew our attention from a group of 14 bacterial isolates during the primary screening for being potentially active against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 9341, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231 and exhibited the broad-spectrum activity against phytopathogenic fungi. In addition, COPS strain showed production of protease, lipase, and esterase in solid media, and its natural product extract showed potent inhibition against fungal plant pathogens, such as Moniliophthora perniciosa, whose antagonism index (89.32%) exceeded the positive control (74.17%), whereas Sclerotinia sclerotiorum and Ceratocystis paradoxa showed high percentages of inhibition (85.53% and 82.69%, respectively). COPS crude extract also significantly inhibited S. epidermidis ATCC 35984, E. faecium ATCC 700221 (MIC values of 32 µg/mL for both), E. faecalis ATCC 29212 (64 µg/mL), and S. aureus ATCC 25923 (128 µg/mL). We observed moderate antagonistic activity against A. baumannii ATCC 19606 and E. coli ATCC 25922 (both at 512 µg/mL), as well as potent cytotoxic effects on Leishmania infantum and Leishmania major promastigote forms with 78.25% and 57.30% inhibition. In conclusion, this study presents for the first time the isolation of an endophytic B. cepacia strain associated with P. paniculata and enough evidence that these plants may be considered a rich source of microbes for the fight against neglected diseases.

Enzymatic potential and biosurfactant production by endophytic fungi from mangrove forest in Southeastern Brazil.

Microbial activity is the main route for cycling mangrove nutrients. In general, microorganisms have abilities to degrade lignocellulosic compounds. Among the biotechnological potential of the microbiota from mangroves, it is noteworthy about endophytic fungi, which can be considered as effective sources of different bioactive compounds. In this sense, thirty (30) endophytic fungi were isolated from mangrove forest sampling Cananeia, SP, Brazil. These microorganisms were analyzed about their enzymatic activities including: lignin peroxidase EC 1.11.1.14, manganese peroxidase EC 1.11.1.13 and laccase EC 1.10.3.2, as well endo-cellulase EC 3.2.1.4 and endo-xylanase EC 3.2.1.8. Besides that, production of bioactive secondary metabolites like biosurfactant and/or bioemulsifier was also investigated. As results, nineteen (19) isolates were selected about their ligninolytic abilities, nine (9) of them about cellulase activity and thirteen (13) showed xylanase abilities. The fungal isolate named as 3(3), characterized as Fusarium sambucinum, showed a prominent lignin peroxidase (42.4 U L-1) and manganese peroxidase (23.6 U L-1) activities. The isolate 63.1, also related to Fusarium sp. genera, was selected about its laccase activity (41.5 U L-1). From all the investigated fungi, the isolate 47(4) Trichoderma camerunense was selected about its cellulolytic and xylanolytic activities, showing 45.23 and 26.09 U mL-1, respectively. The same fungi also showed biosurfactant ability demonstrated by superficial tension decreasing to 38 mN/m. In addition, fifteen (15) fungi exhibited bioemulsifier activity, with E24 values up to 62.8%.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth

ABSTRACT Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000 km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

Screening of tropically derived, multi-trait plant growth- promoting rhizobacteria and evaluation of corn and soybean colonization ability.

The present study assessed the plant growth-promoting (PGP) traits and diversity of culturable rhizobacteria associated with guarana (Paullinia cupana), a typical tropical plant. Ninety-six bacteria were isolated, subjected to biochemical tests, and identified by partial or total 16S rDNA sequencing. Proteobacteria and Firmicutes were the dominant rhizospheric phyla found, and Burkholderia and Bacillus were the most abundant genera. Thirteen strains exhibited the four PGP traits evaluated, and most of them belonged to the genus Burkholderia. Two multi-trait PGP strains, RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria), expressively promoted corn and soybean growth under greenhouse conditions. Compared to the non-inoculated control, increases in corn root dry weight of 247.8 and 136.9% were obtained with RZ2MS9 and RZ2MS16 inoculation, respectively, at 60days after seeding. The dry weights of corn and soybean shoots were significantly higher than those of non-inoculated plants, showing increases of more than 47% for both strains and crops. However, soybean root dry weight did not increased after bacterial inoculation with either strain. The colonization behavior of RZ2MS16 was assessed using GFP-labeling combined with fluorescence microscopy and a cultivation-based approach for quantification. RZ2MS16:gfp was able to colonize the roots and shoots of corn and soybean, revealing an endophytic behavior.

Mangrove endophyte promotes reforestation tree (Acacia polyphylla) growth.

Mangroves are ecosystems located in the transition zone between land and sea that serve as a potential source of biotechnological resources. Brazil's extensive coast contains one of the largest mangrove forests in the world (encompassing an area of 25,000km2 along all the coast). Endophytic bacteria were isolated from the following three plant species: Rhizophora mangle, Laguncularia racemosa and Avicennia nitida. A large number of these isolates, 115 in total, were evaluated for their ability to fix nitrogen and solubilize phosphorous. Bacteria that tested positive for both of these tests were examined further to determine their level of indole acetic acid production. Two strains with high indole acetic acid production were selected for use as inoculants for reforestation trees, and then the growth of the plants was evaluated under field conditions. The bacterium Pseudomonas fluorescens (strain MCR1.10) had a low phosphorus solubilization index, while this index was higher in the other strain used, Enterobacter sp. (strain MCR1.48). We used the reforestation tree Acacia polyphylla. The results indicate that inoculation with the MCR1.48 endophyte increases Acacia polyphylla shoot dry mass, demonstrating that this strain effectively promotes the plant's growth and fitness, which can be used in the seedling production of this tree. Therefore, we successfully screened the biotechnological potential of endophyte isolates from mangrove, with a focus on plant growth promotion, and selected a strain able to provide limited nutrients and hormones for in plant growth.

The diversity of citrus endophytic bacteria and their interactions with Xylella fastidiosa and host plants.

The bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC) and has been associated with important losses in commercial orchards of all sweet orange [Citrus sinensis (L.)] cultivars. The development of this disease depends on the environmental conditions, including the endophytic microbial community associated with the host plant. Previous studies have shown that X. fastidiosa interacts with the endophytic community in xylem vessels as well as in the insect vector, resulting in a lower bacterial population and reduced CVC symptoms. The citrus endophytic bacterium Methylobacterium mesophilicum can trigger X. fastidiosa response in vitro, which results in reduced growth and induction of genes associated with energy production, stress, transport, and motility, indicating that X. fastidiosa has an adaptive response to M. mesophilicum. Although this response may result in reduced CVC symptoms, the colonization rate of the endophytic bacteria should be considered in studies that intend to use this endophyte to suppress CVC disease. Symbiotic control is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace pathogens. Candidate endophytes for symbiotic control of CVC must occupy the xylem of host plants and attach to the precibarium of sharpshooter insects to access the pathogen. In the present review, we focus on interactions between endophytic bacteria from sweet orange plants and X. fastidiosa, especially those that may be candidates for control of CVC.

Endophytic bacterial diversity in the phyllosphere of Amazon Paullinia cupana associated with asymptomatic and symptomatic anthracnose.

Endophytes colonize an ecological niche similar to that of phytopathogens, which make them candidate for disease suppression. Anthracnose is a disease caused by Colletotrichum spp., a phytopathogen that can infect guarana (Paullinia cupana), an important commercial crop in the Brazilian Amazon. We investigated the diversity of endophytic bacteria inhabiting the phyllosphere of asymptomatic and symptomatic anthracnose guarana plants. The PCR-denaturation gradient gel electrophoresis (PCR-DGGE) fingerprints revealed differences in the structure of the evaluated communities. Detailed analysis of endophytic bacteria composition using culture-dependent and 16S rRNA clone libraries revealed the presence of Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria phyla. Firmicutes comprised the majority of isolates in asymptomatic plants (2.40E(-4)). However, cloning and sequencing of 16S rRNA revealed differences at the genus level for Neisseria (1.4E(-4)), Haemophilus (2.1E(-3)) and Arsenophonus (3.6E(-5)) in asymptomatic plants, Aquicella (3.5E(-3)) in symptomatic anthracnose plants, and Pseudomonas (1.1E(-3)), which was mainly identified in asymptomatic plants. In cross-comparisons of the endophytic bacterial communities as a whole, symptomatic anthracnose plants contained higher diversity, as reflected in the Shannon-Weaver and Simpson indices estimation (P < 0.05). Similarly, comparisons using LIBSHUFF and heatmap analysis for the relative abundance of operational taxonomic units (OTUs) showed differences between endophytic bacterial communities. These data are in agreement with the NMSD and ANOSIM analysis of DGGE profiles. Our results suggest that anthracnose can restructure endophytic bacterial communities by selecting certain strains in the phyllosphere of P. cupana. The understanding of these interactions is important for the development of strategies of biocontrol for Colletotrichum.

Species diversity of culturable endophytic fungi from Brazilian mangrove forests.

This study aimed to perform a comparative analysis of the diversity of endophytic fungal communities isolated from the leaves and branches of Rhizophora mangle, Avicennia schaueriana and Laguncularia racemosa trees inhabiting two mangroves in the state of São Paulo, Brazil [Cananeia and Bertioga (oil spill-affected and unaffected)] in the summer and winter. Three hundred and forty-three fungi were identified by sequencing the ITS1-5.8S-ITS2 region of rDNA. Differences were observed in the frequencies of fungi isolated from the leaves and branches of these three different plant species sampled from the Bertioga oil spill-affected and the oil-unaffected mangrove sites in the summer and winter; these differences indicate a potential impact on fungal diversity in the study area due to the oil spill. The molecular identification of the fungi showed that the fungal community associated with these mangroves is composed of at least 34 different genera, the most frequent of which were Diaporthe, Colletotrichum, Fusarium, Trichoderma and Xylaria. The Shannon and the Chao1 indices [H'(95 %) = 4.00, H'(97 %) = 4.22, Chao1(95 %) = 204 and Chao1(97 %) = 603] indicated that the mangrove fungal community possesses a vast diversity and richness of endophytic fungi. The data generated in this study revealed a large reservoir of fungal genetic diversity inhabiting these Brazilian mangrove forests and highlighted substantial differences between the fungal communities associated with distinct plant tissues, plant species, impacted sites and sampling seasons.

Endophytic Methylobacterium extorquens expresses a heterologous ß-1,4-endoglucanase A (EglA) in Catharanthus roseus seedlings, a model host plant for Xylella fastidiosa.

Based on the premise of symbiotic control, we genetically modified the citrus endophytic bacterium Methylobacterium extorquens, strain AR1.6/2, and evaluated its capacity to colonize a model plant and its interaction with Xylella fastidiosa, the causative agent of Citrus Variegated Chlorosis (CVC). AR1.6/2 was genetically transformed to express heterologous GFP (Green Fluorescent Protein) and an endoglucanase A (EglA), generating the strains ARGFP and AREglA, respectively. By fluorescence microscopy, it was shown that ARGFP was able to colonize xylem vessels of the Catharanthus roseus seedlings. Using scanning electron microscopy, it was observed that AREglA and X. fastidiosa may co-inhabit the C. roseus vessels. M. extorquens was observed in the xylem with the phytopathogen X. fastidiosa, and appeared to cause a decrease in biofilm formation. AREglA stimulated the production of resistance protein, catalase, in the inoculated plants. This paper reports the successful transformation of AR1.6/2 to generate two different strains with a different gene each, and also indicates that AREglA and X. fastidiosa could interact inside the host plant, suggesting a possible strategy for the symbiotic control of CVC disease. Our results provide an enhanced understanding of the M. extorquens-X. fastidiosa interaction, suggesting the application of AR1.6/2 as an agent of symbiotic control.

The diversity of endophytic methylotrophic bacteria in an oil-contaminated and an oil-free mangrove ecosystem and their tolerance to heavy metals.

Methylobacterium strains were isolated from mangrove samples collected in Bertioga, SP, Brazil, from locations either contaminated or uncontaminated by oil spills. The tolerances of the strains to different heavy metals were assessed by exposing them to different concentrations of cadmium, lead, and arsenic (0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM). Additionally, the genetic diversity of Methylobacterium spp. was determined by sequence analysis of the 16S rRNA genes. The isolates from the contaminated locations were grouped, suggesting that oil can select for microorganisms that tolerate oil components and can change the methylotrophic bacterial community. Cadmium is the most toxic heavy metal assessed in this work, followed by arsenic and lead, and two isolates of Methylobacterium were found to be tolerant to all three metals. These isolates have the potential to bioremediate mangrove environments contaminated by oil spills by immobilizing the heavy metals present in the oil.

Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy.

Endophytes are microorganisms that colonize plant tissues internally without causing harm to the host. Despite the increasing number of studies on sweet orange pathogens and endophytes, yeast has not been described as a sweet orange endophyte. In the present study, endophytic yeasts were isolated from sweet orange plants and identified by sequencing of internal transcribed spacer (ITS) rRNA. Plants sampled from four different sites in the state of São Paulo, Brazil exhibited different levels of CVC (citrus variegated chlorosis) development. Three citrus endophytic yeasts (CEYs), chosen as representative examples of the isolates observed, were identified as Rhodotorula mucilaginosa, Pichia guilliermondii and Cryptococcus flavescens. These strains were inoculated into axenic Citrus sinensis seedlings. After 45 days, endophytes were re-isolated in populations ranging from 10(6) to 10(9) CFU/g of plant tissue, but, in spite of the high concentrations of yeast cells, no disease symptoms were observed. Colonized plant material was examined by scanning electron microscopy (SEM), and yeast cells were found mainly in the stomata and xylem of plants, reinforcing their endophytic nature. P. guilliermondii was isolated primarily from plants colonized by the causal agent of CVC, Xylella fastidiosa. The supernatant from a culture of P. guilliermondii increased the in vitro growth of X. fastidiosa, suggesting that the yeast could assist in the establishment of this pathogen in its host plant and, therefore, contribute to the development of disease symptoms.

Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for paratransgenic control strategy of citrus variegated chlorosis.

Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.

Diversity of endophytic bacteria from Eucalyptus species seeds and colonization of seedlings by Pantoea agglomerans.

The diversity and beneficial characteristics of endophytic microorganisms have been studied in several host plants. However, information regarding naturally occurring seed-associated endophytes and vertical transmission among different life-history stages of hosts is limited. Endophytic bacteria were isolated from seeds and seedlings of 10 Eucalyptus species and two hybrids. The results showed that endophytic bacteria, such as Bacillus, Enterococcus, Paenibacillus and Methylobacterium, are vertically transferred from seeds to seedlings. In addition, the endophytic bacterium Pantoea agglomerans was tagged with the gfp gene, inoculated into seeds and further reisolated from seedlings. These results suggested a novel approach to change the profile of the plants, where the bacterium is a delivery vehicle for desired traits. This is the first report of an endophytic bacterial community residing in Eucalyptus seeds and the transmission of these bacteria from seeds to seedlings. The bacterial species reported in this work have been described as providing benefits to host plants. Therefore, we suggest that endophytic bacteria can be transmitted vertically from seeds to seedlings, assuring the support of the bacterial community in the host plant.

Bioassay assessment of metarhizium anisopliae (metchnikoff) sorokin (deuteromycota: hyphomycetes) against Oncometopia facialis (signoret) (hemiptera: cicadellidae)

Citrus Variegated Chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by Xylella fastidiosa and transmitted by sharpshooter insects. In this study, the efficacy of the fungus Metarhizium anisopliae in controlling the sharpshooter Oncometopia facialis was studied by bioassay conditions. In the bioassay, insects were sprayed with a suspension containing 5 X 10(7) conidia mL-1. Adults captured in the field were treated in groups of 10 in a total of 11 replications per treatment. Significant differences between the natural mortality and the mortality of insects treated with the fungus were observed 6 days after inoculations (P<0.05). These significant differences increased until 10 days after treatment. The fungus caused 87.1 percent mortality, with the LT50 varying from 5 to 6 days. The LC50 was 1.2 X 10(6) conidia mL-1, varying from 7.7 X 10(5) to 2 X 10(6) conidia mL-1. The results showed that the sharpshooter O. facialis was susceptible to the entomopathogenic action of M. anisopliae in controlled condition during bioassay.

A Clorose Variegada dos Citros (CVC) é uma doença economicamente importante e destrutiva no Brasil e é causada pela bactéria Xylella fastidiosa e transmitida por insetos vetores tal como Oncometopia facialis. Nesse estudo, a eficácia do fungo Metarhizium anisopliae em controlar o inseto vetor O. facialis foi estudada em condições de bioensaio. Nesse bioensaio, insetos foram pulverizados com uma suspensão de 5 X 10(7) conídio mL-1. Insetos-adultos capturados no campo foram tratados em grupos de 10, em um total de 11 replicatas por tratamento. Diferenças significativas entre a mortalidade natural e a mortalidade dos insetos tratados com o fungo foram observadas em 6 dias após a inoculação (P<0.05). Estas diferenças significativas aumentaram antes do décimo dia após o tratamento. O fungo causou uma mortalidade de 87,1 por cento, com uma LT50 variando entre 5 e 6 dias. A LC50 foi de 1,2 X 10(6) conídio mL-1, variando de 7,7 X 10(5) a 2 X 10(6) conídio mL-1. Estes resultados mostraram que o vetor O. facialis foi susceptível a ação entomopatogênica de M. anisopliae em condições controladas durante o bioensaio.

Bioassay assessment of metarhizium anisopliae (metchnikoff) sorokin (deuteromycota: hyphomycetes) against Oncometopia facialis (signoret) (hemiptera: cicadellidae).

Citrus Variegated Chlorosis (CVC) is an economically important, destructive disease in Brazil and is caused by Xylella fastidiosa and transmitted by sharpshooter insects. In this study, the efficacy of the fungus Metarhizium anisopliae in controlling the sharpshooter Oncometopia facialis was studied by bioassay conditions. In the bioassay, insects were sprayed with a suspension containing 5 X 10(7) conidia mL(-1). Adults captured in the field were treated in groups of 10 in a total of 11 replications per treatment. Significant differences between the natural mortality and the mortality of insects treated with the fungus were observed 6 days after inoculations (P<0.05). These significant differences increased until 10 days after treatment. The fungus caused 87.1% mortality, with the LT50 varying from 5 to 6 days. The LC50 was 1.2 X 10(6) conidia mL(-1), varying from 7.7 X 10(5) to 2 X 10(6) conidia mL(-1). The results showed that the sharpshooter O. facialis was susceptible to the entomopathogenic action of M. anisopliae in controlled condition during bioassay.

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus.

Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be studied.

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria.

Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.

Capillary electrophoresis-mass spectrometry of citrus endophytic bacteria siderophores.

CE-ESI-MS with a liquid sheath interface and IT mass analyzer was used for analysis of siderophores from different strains of Methylobacterium spp. citrus endophyte extracts. Three Methylobacterium strains were investigated according to positive bioassay tests. Bacteria cultures were grown under Fe(III) absence (siderophore producing cultures) and under Fe(III) presence (control cultures). Siderophores were extracted from culture supernatant with polystyrene resins. BGE and sheath-liquid composition were optimized, respectively, in order to assure both, best peak resolution and ESI-MS sensitivity. The best analysis conditions were obtained with 100 mmol/L ammonium bicarbonate at pH 8 as BGE and methanol:H(2)O 25:75 + 0.05% formic acid as sheath liquid. CZE-ESI-MS analysis revealed two possible siderophores, according to bacterium species, presenting M(r) of 1004.3 and 798.3 Da.