Endophytic bacteria are ubiquitous in most plant species, residing latently or actively colonizing plant tissues locally as well as systemically. Several definitions have been proposed for endophytic bacteria; in this review endophytes will be defined as those bacteria that can be isolated from surface-disinfested plant tissue or extracted from within the plant, and that do not visibly harm the plant. While this definition does not include nonextractable endophytic bacteria, it is a practical definition based on experimental limitations and is inclusive of bacterial symbionts, as well as internal plant-colonizing nonpathogenic bacteria with no known beneficial or detrimental effects on colonized plants. Historically, endophytic bacteria have been thought to be weakly virulent plant pathogens but have recently been discovered to have several beneficial effects on host plants, such as plant growth promotion and increased resistance against plant pathogens and parasites. In general, endophytic bacteria originate from the epiphytic bacterial communities of the rhizosphere and phylloplane, as well as from endophyte-infested seeds or planting materials. Besides gaining entrance to plants through natural openings or wounds, endophytic bacteria appear to actively penetrate plant tissues using hydrolytic enzymes like cellulase and pectinase. Since these enzymes are also produced by pathogens, more knowledge on their regulation and expression is needed to distinguish endophytic bacteria from plant pathogens. In general, endophytic bacteria occur at lower population densities than pathogens, and at least some of them do not induce a hypersensitive response in the plant, indicating that they are not recognized by the plant as pathogens. Evolutionarily, endophytes appear to be intermediate between saprophytic bacteria and plant pathogens, but it can only be speculated as to whether they are saprophytes evolving toward pathogens, or are more highly evolved than plant pathogens and conserve protective shelter and nutrient supplies by not killing their host. Overall, the endophytic microfloral community is of dynamic structure and is influenced by biotic and abiotic factors, with the plant itself constituting one of the major influencing factors. Since endophytic bacteria rely on the nutritional supply offered by the plant, any parameter affecting the nutritional status of the plant could consequently affect the endophytic community. This review summarizes part of the work being done on endophytic bacteria, including their methodology, colonization, and establishment in the host plant, as well as their role in plant–microbe interactions. In addition, speculative conclusions are raised on some points to stimulate thought and research on endophytic bacteria.Key words: endophytic bacteria, methods, localization, diversity, biological control.

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.

This paper presents the most common methods for extracting (including Baermann funnel, mistifier, maceration, sieving, elutriation and flotation techniques), processing, examining (handling and fixing) and detecting plant and soil nematodes. Some molecular techniques (e.g. DNA extraction, PCR, sequencing and amplified fragment length polymorphism) available for detection and identification of nematodes are also discussed.

To study the effect of microenvironments on potato-associated bacteria, the abundance and diversity of bacteria isolated from the rhizosphere, phyllosphere, endorhiza, and endosphere of field grown potato was analyzed. Culturable bacteria were obtained after plating on R2A medium. The endophytic populations averaged 10(3) and 10(5) CFU/g (fresh wt.) for the endosphere and endorhiza. respectively, which were lower than those for the ectophytic microenvironments, with 10(5) and 10(7) CFU/g (fresh wt.) for the phyllosphere and rhizosphere, respectively. The composition and richness of bacterial species was microenvironment-dependent. The occurrence and diversity of potato-associated bacteria was additionally monitored by a cultivation-independent approach using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of microenvironment-specific communities. In an approach to measure the antagonistic potential of potato-associated bacteria, a total of 440 bacteria was screened by dual testing for in vitro antagonism towards the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. The proportion of isolates with antagonistic activity was highest for the rhizosphere (10%), followed by the endorhiza (9%), phyllosphere (6%), and endosphere (5%). All 33 fungal antagonists were characterized by testing their in vitro antagonistic mechanisms, including their glucanolytic, chitinolytic, pectinolytic, cellulolytic, and proteolytic activity, and by their BOX-PCR fingerprints. In addition, they were screened for their biocontrol activity against Meloidogyne incognita. Overall, nine isolates belonging to Pseudomonas and Streptomyces species were found to control both fungal pathogens and M. incognita and were therefore considered as promising biological control agents.

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bUnderstanding the interactions of plant-parasitic nematodes with antagonistic soil microbes could provide opportunities for novel crop protection strategies. Three arable soils were investigated for their suppressiveness against the root knot nematode Meloidogyne hapla. For all three soils, M. hapla developed significantly fewer galls, egg masses, and eggs on tomato plants in unsterilized than in sterilized infested soil. Egg numbers were reduced by up to 93%. This suggested suppression by soil microbial communities. The soils significantly differed in the composition of microbial communities and in the suppressiveness to M. hapla. To identify microorganisms interacting with M. hapla in soil, second-stage juveniles (J2) baited in the test soil were cultivation independently analyzed for attached microbes. PCR-denaturing gradient gel electrophoresis of fungal ITS or 16S rRNA genes of bacteria and bacterial groups from nematode and soil samples was performed, and DNA sequences from J2-associated bands were determined. The fingerprints showed many species that were abundant on J2 but not in the surrounding soil, especially in fungal profiles. Fungi associated with J2 from all three soils were related to the genera Davidiella and Rhizophydium, while the genera Eurotium, Ganoderma, and Cylindrocarpon were specific for the most suppressive soil. Among the 20 highly abundant operational taxonomic units of bacteria specific for J2 in suppressive soil, six were closely related to infectious species such as Shigella spp., whereas the most abundant were Malikia spinosa and Rothia amarae, as determined by 16S rRNA amplicon pyrosequencing. In conclusion, a diverse microflora specifically adhered to J2 of M. hapla in soil and presumably affected female fecundity.

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