Actinobacterial community and diversity in rhizosphere soils of Aquilaria crassna Pierre ex Lec assessed by RT-PCR and PCR-DGGE

The actinobacterial community in rhizospheres of eaglewood (Aquilaria crassna Pierre ex Lec) was analyzed using culture-independent methods of RT-PCR and PCR DGGE of 16S rRNA gene. We conducted the experiments to investigate the difference in diversity and community structure of actinobacteria with respect to sampling sites and seasons and to determine effect of plant species on selection of rhizosphere community from different sampling sites. Total genomic DNA and RNA were extracted from rhizosphere soils collected from two plantations in Phetchabun province and one plantation in each Nakhonnayok province, Rayong province and Chiang Mai province of Thailand during dry and rainy seasons. The UPGMA dendrogram generated from DGGE fingerprints showed that the actinobacterial community was separated corresponding to sampling sites, suggesting sampling sites effect. The shift in community and diversity between two seasons was detected in all sampling sites. RNA-based analyses showed that several actinobacterial groups appeared to be ubiquitous but different in metabolic activity in different environments. Species diversity (S) and simple indexes (I) indicate the increase in species diversity of actinobacteria from all sampling sites in rainy season. Cloning and sequencing of 16S rRNA gene fragments obtained from DGGE bands revealed that 14 of 40 dominant species of actinobacteria in the rhizospheres of this plant belonged to uncultured actinobacteria. Besides the uncultured actinobacteria, Nocardioides sp., Streptomyces sp., Mycobacterium sp., Rhodococcus sp. and Actinoplanes sp. were indentified and frequently found more than other genera.     

Endophytic actinomycetes isolated from Aquilaria crassna Pierre ex Lec and screening of plant growth promoters production

A total of 10 endophytic actinomycete strains were successfully isolated from healthy shoots and roots of Aquilaria crassna Pierre ex Lec (eaglewood). Analysis of 16S rDNA sequencing of those isolates showed that they belong to members of the genera Streptomyces (2 isolates), Nonomuraea (1 isolate), Actinomadura (1 isolate), Pseudonocardia (1 isolate) and Nocardia (3 isolates). The remaining 2 isolates were unidentified. All of isolates produced the amount of indole-3-acetic acid (IAA) and ammonia ranging between 9.85 ± 0.31 to 15.14 ± 0.22 μg ml^?1 and 2 to 60 mg ml^?1, respectively. Among 10 isolates tested, the amount of hydroxamate-type siderophore produced by 2 isolates was undetectable. While the remaining 8 isolates produced the amount of hydroxamate-type ranging between 3.21 ± 0.12 and 39.30 ± 0.40 μg ml^?1. Also, catechols-type siderophore produced by 9 isolates was undetectable. Actinomadura glauciflava is only one isolate that produced catechols-type 4.12 ± 0.90 μg ml^?1. In addition, 10 endophytic actinomycetes showed protease activity ranging from undetectable to 8.16 ± 0.15 unit ml^?1. Genetic relatedness amongst these isolates was determined base on Random amplified polymorphic DNA (RAPD) and Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC PCR). Both methodologies generated specific patterns corresponding to particular genotypes. RAPD fingerprinting proved to be slightly more discriminatory than ERIC PCR. This study is the first published report that actinomycetes can be isolated as endophytes within this plant. It is also the first published report that endophytic actinomycetes are capable of producing IAA and siderophores.     

Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.     

Effect of Microbial Inoculants on the Indigenous Actinobacterial Endophyte Population in the Roots of Wheat as Determined by Terminal Restriction Fragment Length Polymorphism

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.     

Rational Approaches to Improving the Isolation of Endophytic Actinobacteria from Australian Native Trees

In recent years, new actinobacterial species have been isolated as endophytes of plants and shrubs and are sought after both for their role as potential producers of new drug candidates for the pharmaceutical industry and as biocontrol inoculants for sustainable agriculture. Molecular-based approaches to the study of microbial ecology generally reveal a broader microbial diversity than can be obtained by cultivation methods. This study aimed to improve the success of isolating individual members of the actinobacterial population as pure cultures as well as improving the ability to characterise the large numbers obtained in pure culture. To achieve this objective, our study successfully employed rational and holistic approaches including the use of isolation media with low concentrations of nutrients normally available to the microorganism in the plant, plating larger quantities of plant sample, incubating isolation plates for up to 16 weeks, excising colonies when they are visible and choosing Australian endemic trees as the source of the actinobacteria. A hierarchy of polyphasic methods based on culture morphology, amplified 16S rRNA gene restriction analysis and limited sequencing was used to classify all 576 actinobacterial isolates from leaf, stem and root samples of two eucalypts: a Grey Box and Red Gum, a native apricot tree and a native pine tree. The classification revealed that, in addition to 413 Streptomyces spp., isolates belonged to 16 other actinobacterial genera: Actinomadura (two strains), Actinomycetospora (six), Actinopolymorpha (two), Amycolatopsis (six), Gordonia (one), Kribbella (25), Micromonospora (six), Nocardia (ten), Nocardioides (11), Nocardiopsis (one), Nonomuraea (one), Polymorphospora (two), Promicromonospora (51), Pseudonocardia (36), Williamsia (two) and a novel genus Flindersiella (one). In order to prove novelty, 12 strains were characterised fully to the species level based on polyphasic taxonomy. One strain represented a novel genus in the family Nocardioides, and the other 11 strains were accepted as novel species. In summary, the holistic isolation strategies were successful in obtaining significant culturable actinobacterial diversity within Australian native trees that includes rare and novel species.     

Diversity and antibacterial activity of culturable actinobacteria isolated from five species of the South China Sea gorgonian corals

This study describes the diversity and antibacterial activity of culturable actinobacteria isolated from five species of gorgonian corals (Echinogorgia aurantiaca, Melitodes squamata, Muricella flexuosa, Subergorgia suberosa, and Verrucella umbraculum) collected in shallow water of the South China Sea. A total of 123 actinobacterial isolates were recovered using ten different isolation media, and assigned to 11 genera, including Streptomyces and Micromonospora as the dominant genera, followed by Nocardia, Verrucosispora, Nocardiopsis, Rhodococcus, Pseudonocardia, Agrococcus, Saccharomonospora, Saccharopolyspora and Dietzia. Comparable analysis indicated that the numbers of actinobacterial genera and isolates from the five gorgonian coral species varied significantly. It was found that 72 isolates displayed antibacterial activity against at least one indicator bacterium, and the antibacterial strains isolated from different gorgonians had almost the same proportion (~50 %). These results provide direct evidence for the hypotheses that gorgonian coral species contain large and diverse communities of actinobacteria, and suggest that many gorgonian-associated actinobacteria could produce some antibacterial agents to protect their hosts against pathogens. To our knowledge, this is the first report about the diversity of culturable actinobacteria isolated from gorgonian corals.     

Impact of Agricultural Practices on the Zea mays L. Endophytic Community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

Illumina-Based Sequencing Analysis Directed Selection for Actinobacterial Probiotic Candidates for Banana Plants

As potential probiotic candidates, plant vertically transmitted actinobacteria are beneficial to growth and health of host plants. New methods to isolate the actinobacterial taxa with low growth rates should be developed. Based on the actinobacterial population information, the probiotic actinobacterial taxa could be directly isolated from healthy banana shoot tips. However, actinobacterial DNAs with high GC contents could bias estimates of actinobacteria by PCR. In the study, two amplicon sequencing strategies were adopted to elucidate the endophytic actinobacterial community of banana plants. More than 92.5% bacterial OTUs were affiliated with actinobacteria by these two strategies, and total 14,289 actinobacterial OTUs with above 97% similarity were detected in banana shoot tips. Although the libraries generated by the two strategies differed in the abundance of some genera, Mycobacterium and Nocardia dominated both libraries and most actinobacterial taxa were overlapped. Higher phylogenetic resolution actinobacteriome of banana plants was successfully established. Based on the endophytic actinobacterial community information, the streptomycetes were isolated from shoot tips. Pot experiments illustrated that the strain could promote banana plantlet growth and elevate resistance to Fusarium oxysporum f. sp. cubense (FOC) under FOC infested soils. The results suggested that the selection for probiotic agents based on actinobacteriome analysis is reliable and feasible compared with present greenhouse selection.     

Endophytic bacteria in long-term in vitro cultivated “axenic” pineapple microplants revealed by PCR–DGGE

In vitro propagated plants are believed to be free of microbes. However, after 5 years of in vitro culture of pineapple plants, without evidence of microbial contamination, the use of culture-independent molecular approach [classifying heterogeneous nucleic acids amplified via universal and specific 16S rRNA gene by polymerase chain reaction (PCR)], and further analysis by denaturing gradient gel electrophoresis (DGGE) revealed endophytic bacteria in roots, young and mature leaves of such plants. The amplification of 16S rRNA gene (Bacteria domain) with the exclusion of the plant chloroplast DNA interference, confirmed the presence of bacterial DNA, from endophytic microorganisms within microplant tissues. PCR–DGGE analysis revealed clear differences on bacterial communities depending on plant organ. Group-specific DGGE analyses also indicated differences in the structures of Actinobacteria, Alphaproteobacteria and Betaproteobacteria communities in each part of plants. The results suggest the occurrence of a succession of bacterial communities colonizing actively the microplants organs. This study is the first report that brings together evidences that pineapple microplants, previously considered axenic, harbor an endophytic bacterial community encompassing members of Actinobacteria, Alphaproteobacteria and Betaproteobacteria group which is responsive to differences in organs due to plant development.     

小蓬竹根际土壤微生物及内生真菌多样性分析

为了解喀斯特典型物种-小蓬竹根际土壤微生物及不同部位内生真菌多样性,采用沿等高线等距离取样法采集小蓬竹根际土壤及健康植株,通过可培养对根际土微生物及内生菌进行分离,利用分子技术对其进行鉴定,根据鉴定结果构建系统发育树,并计算小蓬竹根际土壤微生物和根茎叶内生真菌多样性。结果如下：（1）共从根际土壤、根、茎、叶分离得到139个真菌菌株,隶属于27属,其中根际土壤分离得到34个真菌菌株隶属于12属,根部分离得到的63个内生真菌菌株隶属于17个属,茎部分离得到的14个内生真菌菌株隶属于8个属,叶部分离得到28个内生真菌菌株隶属于9个属;（2）根际土壤共分离得到41株细菌菌株,隶属于7个属26个种,20株放线菌菌株,隶属于1属15种;从Shannon-Wiener多样性指数、均匀度指数、Simpson指数排序来看,真菌主要表现为根 > 根际土壤 > 茎 > 叶,细菌和放线菌多样性均较低。（3）按层次聚类分析可分别将真菌、细菌、放线菌聚为3支。小蓬竹根际土壤、根、茎和叶具有丰富的微生物多样性,不同部位菌群组成存在差异性（P<0.05）,且存在以假单胞菌属、芽孢杆菌属等为优势属的抗盐耐旱菌群,这有助于揭示小蓬竹对喀斯特生境的适应性,以及为微生物-植物群落之间相互关系提供一定基础数据,为后期寻找小蓬竹相关耐性功能菌奠定基础。     

Isolation of rhizospheric and roots endophytic actinomycetes from Leguminosae plant and their activities to inhibit soybean pathogen, Xanthomonas campestris pv. glycine

In this study, actinomycetes from roots and rhizospheric soils of leguminous plants were isolated using starch casein agar supplemented with antifungal and antibacterial antibiotics. Three hundred and seventeen actinomycetes were isolated with 77 isolates obtained from plant roots and 240 isolates from rhizospheric soils. Analysis of whole-organism hydrolysates showed that 289 strains were rich in the LL-isomer of diaminopimelic acid, a result consistent with their assignment to the streptomycetes. The remaining 28 strains were assigned to non-streptomycetes based on the presence of meso-isomer of diaminopimelic acid in cell wall. Sixty-four isolates (20.2 %) showed antagonistic activity against soybean pathogen Xanthomonas campestris pv. glycine by agar overlay method. Isolate RM 365 showed the highest activity with an inhibition ratio of 3.79, with no inhibitory activity on the growth of Rhizobium japonicum TISTR 079, Rhizobium sp. TISTR 061 and Rhizobium sp. TISTR 063. The 16S rRNA gene sequence analysis revealed that isolate RM 365 shared 99.28 % similarity to Streptomyces caeruleatus GIMN4^T (GQ329712). In addition, isolates which contained meso-DAP were also identified by 16S rRNA gene sequence analysis. The results showed that they were members of the genus Amycolatopsis, Isoptericola, Micromonospora, Microbispora, Nocardia, Nonomuraea, Promicromonospora and Pseudonocardia.     

Diversity and novelty of actinobacteria in Arctic marine sediments

The actinobacterial diversity of Arctic marine sediments was investigated using culture-dependent and culture-independent approaches. A total of 152 strains were isolated from seven different media; 18 isolates were selected for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 18 isolates belonged to a potential novel genus and 10 known genera including Actinotalea, Arthrobacter, Brachybacterium, Brevibacterium, Kocuria, Kytococcus, Microbacterium, Micrococcus, Mycobacterium, and Pseudonocardia. Subsequently, 172 rDNA clones were selected by restriction fragment length polymorphism analysis from 692 positive clones within four actinobacteria-specific 16S rDNA libraries of Arctic marine sediments, and then these 172 clones were sequenced. In total, 67 phylotypes were clustered in 11 known genera of actinobacteria including Agrococcus, Cellulomonas, Demequina, Iamia, Ilumatobacter, Janibacter, Kocuria, Microbacterium, Phycicoccus, Propionibacterium, and Pseudonocardia, along with other, unidentified actinobacterial clones. Based on the detection of a substantial number of uncultured phylotypes showing low BLAST identities (<95 %), this study confirms that Arctic marine environments harbour highly diverse actinobacterial communities, many of which appear to be novel, uncultured species.     

Microbacterium endophyticum sp. nov. and Microbacterium halimionae sp. nov., endophytes isolated from the salt-marsh plant Halimione portulacoides and emended description of the genus Microbacterium

Fifteen actinobacterial isolates retrieved from the endophytic community of the salt-marsh plant Halimione portulacoides were characterised in this study. ERIC-PCR fingerprinting analysis divided these isolates into two groups represented by strains PA15^T and PA36^T, respectively.     

川楝内生放线菌的分离及多样性研究

【目的】分离四川省各个地区川楝内生放线菌并研究其物种多样性。【方法】应用7种选择性分离培养基分离样品根、茎、叶、树皮和果实中的内生放线菌,采用16SrRNA基因RFLP分析代表菌株多样性。【结果】研究共获得403株内生放线菌。不同地点、不同植株部位、不同培养基分离得到的内生放线菌数目均有差异。广元采集的样品分离得到的数目最多,为86株;最少的是绵阳,仅有12株。从植物表皮中分离到148株放线菌,占获得菌株总数的36.7%;而从果中分离到31株,仅占获得菌株总数的7.6%;虽然从根部分离到的数量也很少,但是其出菌率却是最高的。5号和3号培养基的分离效果最为理想。16S rRNA基因RFLP分析结果显示所有供试菌株在68%的相似性上聚在一起,在84%的相似水平上分成了10个遗传类型。代表菌株的16SrRNA基因序列测定及系统发育分析结果表明:分离得到的放线菌包括4个属,分别是链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。其中,链霉菌是优势类群,占代表菌株数目的比例高达91%,而稀有放线菌的比例只有9%。【结论】研究发现的川楝内生放线菌主要属于链霉菌属(Streptomyces)、北里孢菌属(Kitasatospora)、节杆菌属(Arthrobacter)、克里布所菌属(Kribbella)。     

Impact of agricultural practices on the Zea mays L. endophytic community

Agricultural practices are known to alter bulk soil microbial communities, but little is known about the effect of such practices on the plant endophytic community. We assessed the influence of long-term applications (20 years) of herbicides and different fertilizer types on the endophytic community of maize plants grown in different field experiments. Nested PCR-denaturing gradient gel electrophoresis (DGGE) analyses targeting general bacteria, type I or II methanotrophs, actinomycetes, and general fungi were used to fingerprint the endophytic community in the roots of Zea mays L. Low intraplant variability (reproducible DGGE patterns) was observed for the bacterial, type I methanotroph, and fungal communities, whereas the patterns for endophytic actinomycetes exhibited high intraplant variability. No endophytic amplification product was obtained for type II methanotrophs. Cluster and stability analysis of the endophytic type I methanotroph patterns differentiated maize plants cultivated by using mineral fertilizer from plants cultivated by using organic fertilizer with a 100% success rate. In addition, lower methanotroph richness was observed for mineral-fertilized plants than for organically fertilized plants. The use of herbicides could not be traced by fingerprinting the endophytic type I methanotrophs or by evaluating any other endophytic microbial group. Our results indicate that the effect of agrochemicals is not limited to the bulk microbial community but also includes the root endophytic community. It is not clear if this effect is due to a direct effect on the root endophytic community or is due to changes in the bulk community, which are then reflected in the root endophytic community.     

Effect of microbial inoculants on the indigenous actinobacterial endophyte population in the roots of wheat as determined by terminal restriction fragment length polymorphism

The effect of single actinobacterial endophyte seed inoculants and a mixed microbial soil inoculant on the indigenous endophytic actinobacterial population in wheat roots was investigated by using the molecular technique terminal restriction fragment length polymorphism (T-RFLP). Wheat was cultivated either from seeds coated with the spores of single pure actinobacterial endophytes of Microbispora sp. strain EN2, Streptomyces sp. strain EN27, and Nocardioides albus EN46 or from untreated seeds sown in soil with and without a commercial mixed microbial soil inoculant. The endophytic actinobacterial population within the roots of 6-week-old wheat plants was assessed by T-RFLP. Colonization of the wheat roots by the inoculated actinobacterial endophytes was detected by T-RFLP, as were 28 to 42 indigenous actinobacterial genera present in the inoculated and uninoculated plants. The presence of the commercial mixed inoculant in the soil reduced the endophytic actinobacterial diversity from 40 genera to 21 genera and reduced the detectable root colonization by approximately half. The results indicate that the addition of a nonadapted microbial inoculum to the soil disrupted the natural actinobacterial endophyte population, reducing diversity and colonization levels. This was in contrast to the addition of a single actinobacterial endophyte to the wheat plant, where the increase in colonization level could be confirmed even though the indigenous endophyte population was not adversely affected.     

Bacterial Communities Associated with the Leaves and the Roots of Arabidopsis thaliana

Diverse communities of bacteria inhabit plant leaves and roots and those bacteria play a crucial role for plant health and growth. Arabidopsis thaliana is an important model to study plant pathogen interactions, but little is known about its associated bacterial community under natural conditions. We used 454 pyrosequencing to characterize the bacterial communities associated with the roots and the leaves of wild A. thaliana collected at 4 sites; we further compared communities on the outside of the plants with communities in the endophytic compartments. We found that the most heavily sequenced bacteria in A. thaliana associated community are related to culturable species. Proteobacteria, Actinobacteria, and Bacteroidetes are the most abundant phyla in both leaf and root samples. At the genus level, sequences of Massilia and Flavobacterium are prevalent in both samples. Organ (leaf vs root) and habitat (epiphytes vs endophytes) structure the community. In the roots, richness is higher in the epiphytic communities compared to the endophytic compartment (P = 0.024), while the reverse is true for the leaves (P = 0.032). Interestingly, leaf and root endophytic compartments do not differ in richness, diversity and evenness, while they differ in community composition (P = 0.001). The results show that although the communities associated with leaves and roots share many bacterial species, the associated communities differ in structure.     

Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic activities in vitro

The populations of endophytic fungi and actinomycetes from four rice cultivars in the Panyu district (Site 1) and Wushan district (Site 2) in Guangdong province, South China, were studied. The preponderant endophytic fungi and actinomycetes isolated belonged to Fusarium and Streptomyces respectively. The incidence of Streptomycetes griseofuscus ranged from 36.1 to 69% out of all the different rice cultivars from the two sites. It is the commonest population of endophytic actinomycetes, and constituted the greatest part of all the antagonistic communities. The distributions of endophytic fungi and actinomycetes in roots and leaves were different, endophytic fungi from leaves were diverse, some were organ-specific. More diverse endophytic actinomycetes were isolated from roots than from leaves. The endophytic fungi isolated from rice in Site 2 were more diverse than that in Site 1. The diversity of the endophytic actinomycetes, however, was less than that in Site 1. Acid soil in Site 2 is ideal for the growth and colonization of fungi while the alkaline soil in Site 1 is better for the growth and colonization of actinomycetes. The results suggested that differences in the chemical composition of soil could influence the endophytic microbial communities of rice plants. The endophytic fungi and actinomycetes isolated from poor-growing seedlings and susceptible rice cultivars were more abundant than that the disease-resistant counterparts. In the dual culture and activity detection of the metabolites, 41.2% of all the isolated endophytic fungi showed antagonism to rice pathogens. Fifty percent of all the isolated endophytic actinomycetes were antagonistic to those pathogens. The percentage of Streptomyces griseofuscus and hygroscopicus reached 55.4 and 21.4% of all the active actinomycetes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA clones

The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.