Colonization of rice and Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) larvae by genetically modified endophytic Methylobacterium mesophilicum

The colonization of Spodoptera frugiperda J.E. Smith larvae and rice seedlings by genetically modified endophytic bacterium Methylobacterium mesophilicum, and also the possible transfer of this bacterium to inside the larva's body during seedlings consumption were studied. The data obtained by bacterial reisolation and fluorescence microscopy showed that the bacterium colonized the rice seedlings, the larva's body and that the endophytic bacteria present in seedlings could be acquired by the larvae. In that way, the transference of endophytic bacterium from plants to insect can be a new and important strategy to insect control using engineered microorganisms.CNPq National Council of Researc

Isolation and enzyme bioprospection of endopytic bacteria associated with plants of Brazilian mangrove ecosystem

The mangrove ecosystem is a coastal tropical biome located in the transition zone between land and sea that is characterized by periodic flooding, which confers unique and specific environmental conditions on this biome. In these ecosystems, the vegetation is dominated by a particular group of plant species that provide a unique environment harboring diverse groups of microorganisms, including the endophytic microorganisms that are the focus of this study. Because of their intimate association with plants, endophytic microorganisms could be explored for biotechnologically significant products, such as enzymes, proteins, antibiotics and others. Here, we isolated endophytic microorganisms from two mangrove species, Rhizophora mangle and Avicennia nitida, that are found in streams in two mangrove systems in Bertioga and Cananéia, Brazil. Bacillus was the most frequently isolated genus, comprising 42% of the species isolated from Cananéia and 28% of the species from Bertioga. However, other common endophytic genera such as Pantoea, Curtobacterium and Enterobacter were also found. After identifying the isolates, the bacterial communities were evaluated for enzyme production. Protease activity was observed in 75% of the isolates, while endoglucanase activity occurred in 62% of the isolates. Bacillus showed the highest activity rates for amylase and esterase and endoglucanase. To our knowledge, this is the first reported diversity analysis performed on endophytic bacteria obtained from the branches of mangrove trees and the first overview of the specific enzymes produced by different bacterial genera. This work contributes to our knowledge of the microorganisms and enzymes present in mangrove ecosystems

Detection of the bacterium, Xylella fastidiosa, in saliva of glassy-winged sharpshooter, Homalodisca vitripennis

Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), the glassy-winged sharpshooter, is one of the most important vectors of the bacterium, Xylella fastidiosa subsp. piercei (Xanthomonadales: Xanthomonadaceae) that causes Pierce’s Disease in grapevines in California. In the present study we report a new method for studying pathogen transmission or probing behavior of H. vitripennis. When confined, H. vitripennis attempt to probe the surface of sterile containers 48 hours post-acquisition of X. f. piercei. The saliva deposited during attempted feeding probes was found to contain X. f. piercei. We observed no correlation between X. f. piercei titers in the foregut of H. vitripennis that fed on Xylella-infected grapevines and the presence of this bacterium in the deposited saliva. The infection rate after a 48 h post-acquisition feeding on healthy citrus and grapevines was observed to be 77% for H. vitripennis that fed on grapevines and 81% for H. vitripennis that fed on citrus, with no difference in the number of positive probing sites from H. vitripennis that fed on either grapevine or citrus. This method is amenable for individual assessment of X. f. piercei-infectivity, with samples less likely to be affected by tissue contamination that is usually present in whole body extracts

Diversity and biotechnological potential of plant-associated endophytic bacteria

Endophytes are microorganisms that inhabit the interior of plant tissues without harming their hosts. This definition includes internal colonists with apparently neutral behavior, symbionts and bacteria that migrate between the surface and the inside of the plant during their endophytic phase. The utilization of endophytes for biotechnological purposes has increased recently, especially in pest and disease control and in plant growth promotion. Endophytic bacteria promote plant growth in three major ways: they synthesize particular compounds that are useful for the plants, facilitate the uptake of certain nutrients from the soil, and control or prevent diseases (biological control). Growth promotion mediated by endophytic bacteria occurs via several mechanisms: the production of vital enzymes; the production of hormones such as auxin (indoleacetic acid [IAA]); symbiotic nitrogen fixation; antagonism against phytopathogens via the production of siderophores, chitinases or antibiotics and the solubilization and mineralization of nutrients, particularly insoluble mineral phosphates. The interactions between endophytes and plants can promote plant health and play a significant role in low-input sustainable agriculture for both food and nonfood crops. An understanding of the mechanisms enabling these endophytes to interact with plants is essential for realizing the biotechnological potential of these microorganisms

Diversity and biotechnological potential of culturable bacteria from Brazilian mangrove sediment

Mangrove ecosystems are environments subject to substantial degradation by anthropogenic activities. Its location, in coastal area, interfacing the continents and the oceans makes it substantially important in the prospection for biotechnological applications. In this study, we assessed the diversity of culturable bacteria present over the seasons at two depths (0-10 and 30-40 cm) in a mangrove sediment and in a transect area from the land to the sea. In total, 238 bacteria were isolated, characterized by Amplified Ribosomal DNA Restriction Analysis (ARDRA) and further identified, by Fatty Acid Methyl Esther (FAME-MIDI), into the orders of Vibrionales, Actinomycetales and Bacillales. Also the ability of the isolates in producing economically important enzymes (amylases, proteases, esterases and lipases) was evaluated and the order Vibrionales was the main enzymatic source.FAPESP/BIOTA[04/13910-6]ISM, fellowship[06/57060-1]PTL and fellowship[07/56360-4]CNP

Isolation of micropropagated strawberry endophytic bacteria and assessment of their potential for plant growth promotion

Twenty endophytic bacteria were isolated from the meristematic tissues of three varieties of strawberry cultivated in vitro, and further identified, by FAME profile, into the genera Bacillus and Sphingopyxis. The strains were also characterized according to indole acetic acid production, phosphate solubilization and potential for plant growth promotion. Results showed that 15 strains produced high levels of IAA and all 20 showed potential for solubilizing inorganic phosphate. Plant growth promotion evaluated under greenhouse conditions revealed the ability of the strains to enhance the root number, length and dry weight and also the leaf number, petiole length and dry weight of the aerial portion. Seven Bacillus spp. strains promoted root development and one strain of Sphingopyxis sp. promoted the development of plant shoots. The plant growth promotion showed to be correlated to IAA production and phosphate solubilization. The data also suggested that bacterial effects could potentially be harnessed to promote plant growth during seedling acclimatization in strawberry.CapesCNP

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.FAPESP/BIOTAFAPESP/BIOTA [04/13910-6, 06/57060-1]CNPqCNP