Expression of the <Emphasis Type="Italic">HEV2.1</Emphasis> gene promoter in transgenic <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

In this article we describe the identification of endophytic bacteria belonging to three groups isolated from shoot tip cultures of banana cv. Grand Naine in a recent study (Thomas et al. 2008) based on partial 16S rRNA gene sequence homology analysis. The first group included banana stocks that displayed obvious colony growth on MS based tissue culture medium during the first in vitro passage. The second group constituted stocks that were tissue index-negative for cultivable bacteria initially but turned index-positive after a few to several (4–8) in vitro passages while the third group formed one sub-stock that turned index-positive after about 18 passages. The organisms belonged to about 20 different genera comprising of α, β, γ-proteobacteria, Gram-positive firmicutes and actinobacteria. Visibly expressing easily cultured organisms during the first in vitro passage included Enterobacter, Klebsiella, Ochrobactrum, Pantoea, Staphylococcus and Bacillus spp. Organisms of second group that were not detected or non-culturable originally constituted Brevundimonas, Methylobacterium, Alcaligenes, Ralstonia, Pseudomonas, Corynebacterium, Microbacterium, Staphylococcus, Oceanobacillus and Bacillus spp. while the third group that turned cultivable after extended in vitro culturing included mostly non-filamentous actinobacteria (Brachybacterium, Brevibacterium, Kocuria and Tetrasphaera spp.). The identification results suggested that the endophytes of second and third groups were not strictly obligate or fastidious microbes but those surviving in viable but-non-culturable (VBNC) state and displaying gradual activation to cultivable form during continuous tissue culturing. Several of the organisms isolated are known as beneficial ones in agriculture while some organisms have possible implications in human health. The use of tissue cultures for isolating uncommon endophytes is discussed. Supply of live bacterial cultures or genetic material for research purpose is subject to their revival from glycerol stocks (as some of the organisms showed poor tolerance) and the requestor obtaining written permission from the Director General, Indian Council of Agricultural Research, New Delhi-110001.     

Infection and Colonization of Sugar Cane and Other Graminaceous Plants by Endophytic Diazotrophs

Agriculturally important grasses such as sugar cane (Saccharum sp.), rice (Oryza sativa), wheat (Triticum aestivum) sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum contain numerous diazotrophic bacteria, such as, Acetobacter diazotrophicus, Herbaspirillum spp., Azospirillum spp. These bacteria do not usually cause disease symptoms in the plants with which they are associated and the more numerous of them, for example, Herbaspirillum spp. and A. diazotrophicus, are obligate or facultative endo-phytes that do not survive well (or at all) in native soil; these are thought to be spread from plant generation to plant generation via seeds, vegetative propagation, dead plant material, and possibly by insect sap feeders. By contrast, Azospirillum spp. are not wholly endophytic but are root-associated, soil-dwelling bacteria that are also often found within plants, probably entering host plants via seeds or via wounds/cracks at lateral root junctions. Endophytic diazotrophs have been isolated from a number of grasses in which significant biological N₂ fixation (BNF) has been demonstrated, particularly Brazilian sugar cane varieties, but also in rice, maize, and sorghum. However, although the endophytic diazotrophs are held to be the causative agents of the observed BNF, direct evidence for this is lacking. Therefore, in this review we examine probable sites of bacterial multiplication and/or BNF within endophyte-containing grasses and discuss these in terms of potential benefits (or not) to both host plants and bacteria. In particular, we examine how potentially large numbers of bacteria, especially Herbaspirillum spp., A. diazotrophicus, and Azospirillum spp., can exist extracellularly within non-specialized (for symbiotic purposes) regions such as xylem vessels and intercellular spaces. The processes of infection and colonization of various grasses (particularly sugar cane) by diazotrophic endophytes are also described, and these are compared with those of important (nondiazotrophic) endophytic sugar cane pathogens such as Clavibacter xyli subsp. xyli and Xanthomonas albilineans.     

Rock-degrading endophytic bacteria in cacti

A plant–bacterium association of the cardon cactus (Pachycereus pringlei) and endophytic bacteria promotes establishment of seedlings and growth on igneous rocks without soil. These bacteria weather several rock types and minerals, unbind significant amounts of useful minerals for plants from the rocks, fix in vitro N₂, produce volatile and non-volatile organic acids, and reduce rock particle size to form mineral soil. This study revealed the presence of large populations of culturable endophytic bacteria inside the seeds extracted from wild plants, from seeds extracted from the guano of bats feeding on cactus fruit, in seedlings growing from these seeds, in the pulp of fruit, and in small, mature wild plants, and are comparable in size to populations of endophytic populations in some agricultural crops. The dominant culturable endophytes were isolates of the genera Bacillus spp., Klebsiella spp., Staphylococcus spp., and Pseudomonas spp. Based on partial sequencing of the 16s rRNA gene, the isolated strains had low similarity to known strains in these genera. However, these strains have higher molecular similarity among endophytes obtained from seeds, endophytes from roots, and some bacterial strains from the rhizoplane. Seedlings developed from seeds with endophytes contain the similar species of endophytes in their shoots, possibly derived from the seeds. This study shows the involvement of endophytic bacteria in rock weathering by cacti in a hot, subtropical desert and their possible contribution to primary colonization of barren rock. This study proposes that cacti capable of acquiring diverse populations of endophytes may give them an evolutionary advantage to gain a foothold on highly uncompromising terrain.     

Ubiquitous presence of normally non-culturable endophytic bacteria in field shoot-tips of banana and their gradual activation to quiescent cultivable form in tissue cultures

Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 10²–10⁵ colony forming units g⁻¹ fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR^+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 10⁵ – 4.0 × 10⁷ colony forming units g⁻¹ tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.     

基于高通量测序分析西藏沙棘根瘤内生菌的多样性

根瘤是微生物侵染植物根部并与之形成的共生结构,这些微生物都可被称为植物内生菌。豆科植物根瘤中的内生菌常常又被称为根瘤菌,而侵染非豆科植物形成根瘤的主要是放线菌弗兰克氏菌,这些非豆科植物又被称为放线菌结瘤植物。西藏沙棘是一种典型的放线菌结瘤植物,由于其分布生境的特殊性,对其根瘤内生菌的研究具有重要的生态意义。对于西藏沙棘根瘤内生菌的研究,培养方法因难以模拟自然条件而不易获得纯培养,高通量测序技术对其多样性的研究提供了便利。因此,本研究以生长在甘肃省天祝县金强河河滩地的西藏沙棘根瘤为材料,采用16S rRNA基因扩增子高通量测序方法,结合OTU分析,对西藏沙棘根瘤内生菌的多样性进行探讨。实验结果表明,西藏沙棘根瘤内生菌具有丰富的多样性,根瘤内的优势属为共生固氮的弗兰克氏菌属（Frankia）,其相对丰度为47.63%,共检测到7个弗兰克氏菌属的OTUs;根瘤内除弗兰克氏菌外,还存在大量的非弗兰克氏菌,共检测到1523个OTUs,隶属于22个门、33个纲、69个目、113个科和202个属,相对丰度排名前9的属中有25个非弗兰克氏菌属的OTUs。该研究也表明,西藏沙棘根瘤内生菌具有丰富的多样性,西藏沙棘根瘤中不仅存在着可共生固氮的弗兰克氏菌,并且还分布着非弗兰克氏菌;在同一根瘤样品中,弗兰克氏菌属还具有不同的物种。本研究不仅拓展了西藏沙棘根瘤内生菌多样性的研究方法,还为同一寄主植物中弗兰克氏菌多样性的研究提供了分析思路。     

Analysis of Endophytic Bacterial Communities of Potato by Plating and Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA Based PCR Fragments

The diversity of endophytic bacterial populations of potato (Solanum tuberosum cv Desirée) was assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates, and direct PCR-DGGE on the basis of DNA extracted from plants. The culturable endophytic bacterial communities detected in potato stem bases as well as in roots were in most cases on the order 10³ to 10⁵ CFU g⁻¹ of fresh plant tissue. Dilution plating revealed that a range of bacterial types dominated these populations. Dominant isolates fell into the α and γ subgroups of the Proteobacteria, as well as in the Flavobacterium/Cytophaga group. Different representatives of the Firmicutes were also found. The most frequently isolated strains (>5% of the total) were characterized as different Pseudomonas spp. (including P. aureofaciens, P. corrugata, and P. putida), Agrobacterium radiobacter, Stenotrophomonas maltophilia, and Flavobacterium resinovorans, using fatty acid methyl ester (FAME) analysis and/or sequencing of their partial 16S ribosomal RNA genes. Other Proteobacteria or Firmicutes were also found, albeit infrequently, and mainly in potato stem tissue. The fate of three putative potato endophytes, Stenotrophomonas maltophilia, Bacillus sp., and Sphingomonas paucimobilis, was monitored following their release into potato plants via injection, via root dipping, or via the soil. Following stem injection, the S. maltophilia and Bacillus inoculants could be tracked over time periods of, respectively, 22 and 1 day(s) by dilution plating as well as via PCR-DGGE. However, only S. maltophilia was able to colonize, and persist in, plant tissue from soil or dipped roots. S. paucimobilis was never recovered from the plant irrespective of the mode of introduction. The diversity of the indigenous bacterial flora associated with potato was then monitored via PCR-DGGE. The patterns obtained revealed the existence of bacterial communities of limited complexity, with communities from potato stems typically differing from those from stem peel and roots. Evidence was obtained for the endophytic occurrence of a range of organisms falling into the α, β, and γ subgroups of the Proteobacteria as well as in the Firmicutes. Several of the sequences found matched those from isolates, suggesting that the molecular evidence reported culturable organisms. However, a number of sequences did not have matching sequences from isolates, suggesting that non-culturable or as-yet-uncultured endophytic organisms were being detected.     

Identification of Diazotrophs in the Culturable Bacterial Community Associated with Roots of <Emphasis Type="Italic">Lasiurus sindicus</Emphasis>, a Perennial Grass of Thar Desert,India

Lasiurus sindicus is a highly nutritive, drought-tolerant, perennial grass that is endemic to the Thar Desert of Rajasthan, India. Analysis of 16S rRNA coding genes of the bacterial isolates enriched in nitrogen-free semisolid medium, from the surface-sterilized roots of L. sindicus, showed predominance of Gram-negative over Gram-positive bacteria. According to comparative sequence analysis of 16S rDNA sequence data, Gram-positive bacteria with low GC content (Staphylococcus warneri and Bacillus sp.) and high GC content (Micrococcus luteus, Microbacterium sp.) were identified. Gram-negative bacteria included Azospirillum sp., Rhizobium sp., Agrobacterium tumefaciens, and Inquilinus limosus (α-proteobacteria); Ralstonia sp., Variovorax paradoxus, and Bordetella petrii (β-proteobacteria); and Pseudomonas pseudoalcaligenes, Stenotrophomonas sp. (γ-proteobacteria). The occurrence of nifH sequences in Azospirillum sp., Rhizobium sp., and P. pseudoalcaligenes showed the possibility of supplying biologically fixed nitrogen by the root-associated diazotrophs to the host plant.     

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface‐sterilized axillary shoot‐buds of papaya ‘Arka Surya’ revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot‐tip‐derived healthy proliferating cultures of three genotypes (‘Arka Surya’, ‘Arka Prabhath’, ‘Red Lady’) with regular monthly subculturing also displayed high bacterial diversity (11–16 phyla, >25 classes, >50 families, >200 genera) about 12–18 months after initial establishment. ‘Arka Surya’ and ‘Red Lady’ cultures bore predominantly Actinobacteria (75–78%) while ‘Arka Prabhath’ showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright‐field direct microscopy on tissue sections and tissue homogenate and epi‐fluorescence microscopy employing bacterial DNA probe SYTO‐9 revealed abundant intracellular bacteria embracing the next‐generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states‐ PICRUSt‐ functional annotation suggested significant operational roles for the bacterial‐biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular‐colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.     

不同水稻组织内生细菌的群落多样性

[目的]为详细了解水稻不同组织内生细菌群落多样性。[方法]对宁粳43号内生细菌的总DNA提取后,采用高通量测序技术对水稻内生细菌的16S rRNA基因进行了序列测定,分析了水稻不同组织部位内生细菌群落结构特征。[结果]叶部共获得内生细菌OTUs 610个,茎部411个,根部174个。物种分类显示,叶部内生细菌种类隶属于22门40纲103目198科399属,其中优势类群是红球菌属（Rhodococcus）和乳酸杆菌属（Lactobacillus）,它们的相对丰度分别为21.00%和9.19%;茎部内生细菌种类隶属于19门31纲85目169科306属,其中优势类群是红球菌属和罗尔斯通菌属（Ralstonia）,它们的相对丰度分别为19.25%和13.52%;根部内生细菌种类隶属于9门19纲44目82科140属,其中优势类群是肠杆菌属（Enterobacter）和埃希氏杆菌属（Escherichia）,它们的相对丰度分别为81.13%和10.89%。根茎叶中相同的OTU有78个,放线菌门（Actinobacteria）与大多数细菌具有相关性。根系内生细菌中具有调控各种代谢网络功能的物种丰度高于茎部和叶部。[结论]不同水稻组织内生细菌具有丰富的群落多样性,其中叶部的内生细菌物种最丰富,根系参与各种代谢调控的细菌丰度最高,各个组织部位的优势菌属各不相同,变形菌门是最重要的水稻内生细菌。     

Effect of Plant Age on Endophytic Bacterial Diversity of Balloon Flower (Platycodon grandiflorum) Root and Their Antimicrobial Activities

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of ‘low G + C gram-positive bacteria’ (LGCGPB) gradually increased 60.0–80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.     

Echinacea purpurea microbiota: bacterial–fungal interactions and the interplay with host and non-host plant species in vitro dual culture

• Important evidence is reported on the antimicrobial and antagonistic properties of bacterial endophytes in Echinacea purpurea and their role in the modulation of plant synthesis of bioactive compounds. Here, endophytic fungi were isolated from E. purpurea, and the dual culture approach was applied to deepen insights into the complex plant–microbiome interaction network.
• In vitro experiments were carried out to evaluate the species specificity of the interaction between host (E. purpurea) and non-host (E. angustifolia and Nicotiana tabacum) plant tissues and bacterial or fungal endophytes isolated from living E. purpurea plants to test interactions between fungal and bacterial endophytes.
• A higher tropism towards plant tissue and growth was observed for both fungal and bacterial isolates compared to controls without plant tissue. The growth of all fungi was significantly inhibited by several bacterial strains that, in turn, were scarcely affected by the presence of fungi. Finally, E. purpurea endophytic bacteria were able to inhibit mycelial growth of the phytopathogen Botrytis cinerea.
• Bacteria and fungi living in symbiosis with wild Echinacea plants interact with each other and could represent a potential source of bioactive compounds and a biocontrol tool.

     

Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.     

Ecology of bacterial endophytes associated with wetland plants growing in textile effluent for pollutant-degradation and plant growth-promotion potentials

In this study, 41 culturable endophytic bacteria were isolated from the roots and shoots of three wetland plants, Typha domingensis, Pistia stratiotes and Eichhornia crassipes, and identified by 16S rRNA gene sequencing. Textile effluent-degrading and plant growth-promoting activities of these endophytes were determined. The analysis of endophytic bacterial communities indicated that plant species had a pronounced effect on endophytic bacterial association and maximum endophytes (56.5%) were associated with T. domingensis. These endophytic bacteria mainly belonged to different species of the genera Bacillus (39%), Microbacterium (12%) and Halomonas (12%). Eight of the 41 strains showing maximum efficiency of textile effluent degradation also exhibited plant growth-promoting activities such as production of indole-3-acetic acid and siderophore, presence of 1-amino-cyclopropane-1-carboxylic acid deaminase, and solubilization of inorganic phosphorous. This is the first study describing the diversity and plant-beneficial characteristics of the textile effluent-degrading endophytic bacteria associated with wetland plants. T. domingensis showed better growth in textile effluent and also hosted maximum number of endophytic bacteria in roots and shoots. The interactions between T. domingensis and its associated endophytic bacteria could be exploited to enhance the efficiency of constructed wetlands during the remediation of industrial effluent.     

Methylobacterium-Induced Endophyte Community Changes Correspond with Protection of Plants against Pathogen Attack

Plant inoculation with endophytic bacteria that normally live inside the plant without harming the host is a highly promising approach for biological disease control. The mechanism of resistance induction by beneficial bacteria is poorly understood, because pathways are only partly known and systemic responses are typically not seen. The innate endophytic community structures change in response to external factors such as inoculation, and bacterial endophytes can exhibit direct or indirect antagonism towards pathogens. Earlier we showed that resistance induction by an endophytic Methylobacterium sp. in potato towards Pectobacterium atrosepticum was dependent on the density of the inoculum, whereas the bacterium itself had no antagonistic activity. To elucidate the role of innate endophyte communities in plant responses, we studied community changes in both in vitro and greenhouse experiments using various combinations of plants, endophyte inoculants, and pathogens. Induction of resistance was studied in several potato (Solanum tuberosum L.) cultivars by Methylobacterium sp. IMBG290 against the pathogens P. atrosepticum, Phytophthora infestans and Pseudomonas syringae pv. tomato DC3000, and in pine (Pinus sylvestris L.) by M. extorquens DSM13060 against Gremmeniella abietina. The capacities of the inoculated endophytic Methylobacterium spp. strains to induce resistance were dependent on the plant cultivar, pathogen, and on the density of Methylobacterium spp. inoculum. Composition of the endophyte community changed in response to inoculation in shoot tissues and correlated with resistance or susceptibility to the disease. Our results demonstrate that endophytic Methylobacterium spp. strains have varying effects on plant disease resistance, which can be modulated through the endophyte community of the host.     

Sanitizing long-term micropropagated grapes from covert and endophytic bacteria and preliminary field testing of plants after 8 years <Emphasis Type="Italic">in vitro</Emphasis>

Summary Micropropagated grape (Vitis vinifera L.) cv. Arka Neelamani cultures showed a decline in root and shoot growth performance after 6–7 yr of continuous in vitro culture. Indexing the culture medium using nutrient agar or 523 bacteriological medium (Viss et al., 1991) revealed covert bacteria in 75–100% cultures. Testing the tissue from different parts of in vitro plantlets on nutrient agar showed bacteria comprising of six or more morphotypes in 100% of root and collar tissue samples but less frequently in stem segments. The shoot tips had the lowest incidence of bacterial association. The whole shoots treated with NaOCl (4% chlorine) or HgCl₂ (0.1%) showed endophytic bacterial survival. Culturing the HgCl₂-treated (5 min) shoot tips on antibiotic overlaid medium (1 ml of 50 mg l⁻¹ gentamycin and/or cefazolin) in culture tubes (150×25 mm) for 1 mo. facilitated the cleansing of cultures with 75% recovery of contaminant-free shoots as monitored through indexing for the next 2 yr. Repeated indexing of medium and tissue from various plant parts during the first two to four subculture cycles following antibiotic treatment was instrumental in reliably identifying clean cultures and preventing bacterial re-emergence. Antibiotic incorporation in the medium was detrimental to grape microcuttings. Bacteria-freed cultures showed 80–100% rooting and a high number of plantlets that could be acclimatized. The plants put in the field after 8 yr of active micropropagation showed some juvenile characteristics initially, which disappeared in 6–8 mo., and the pruned shoots showed flowering and bunch development within 1 yr of field planting. This indicated the feasibility of keeping grape plants in vitro for long periods if covert microbes were eliminated.     

Effect of host genotype on indigenous bacterial endophytes of cotton (Gossypium hirsutum L.)

The purpose of this study was to determine if populations of indigenous bacterial endophytes in seed, stem and root tissue of cotton seedlings are influenced by host genotype. Growth chamber and field experiments were conducted to test the hypothesis that host genotype has an effect on endophytic bacterial populations in seed tissues and the developing cotton seedling. Initially, population densities of bacteria within seed of nine cotton cultivars were very low (i.e., 10 2.0 colony forming units seed^–1). However, after 4 days growth on water agar, population densities within developing radicles increased significantly (log₁₀ 2–5 colony forming units) and significant cultivar differences were found. Significant cultivar differences occurred for populations of endophytic bacteria and the composition of bacterial functional groups differed among cultivars in field-grown seedlings at 5, 8, and 15 days after planting. Differences in the ranking of cultivars occurred for endophytic populations recovered from seed and aseptically and field-grown radicle and seedling tissues. These results suggest that whether originating from seed or from soil, cotton plants are capable of immediately establishing a carrying capacity for communities of endophytic bacteria following seed germination. During germination and development of the seedling, there are genetic and possible morphological/physiological effects that contribute to significant differences in colonization of bacterial endophytes among cotton cultivars.     

Ubiquitous presence of fastidious endophytic bacteria in field shoots and index-negative apparently clean shoot-tip cultures of papaya

This study establishes the widespread prevalence of fastidious or viable but non-culturable endophytic bacteria in field shoots and in unsuspicious shoot-tip cultures of papaya (Carica papaya L.) against the norm of asepsis in vitro. A total of 150 shoot-tips (approximately 10 mm) were inoculated on MS-based culture medium after surface sterilization of field-derived axillary shoots of cv. Surya during November or January (100 and 50, respectively) when 35–50% cultures showed endophytic microbial growth on culture medium. Indexing of apparently clean cultures using bacteriological media helped in detecting and removing additional 14–17% stocks with covert bacteria during the first two passages. The rest of the stocks stayed consistently index-negative during the first eight subculture cycles, but appeared positive in PCR-screening undertaken thereafter employing universal bacterial 16S rRNA gene primers indicating the association of non-cultivable bacteria. Direct sequencing of the PCR product yielded overlapping nucleotide data signifying mixed template or the presence of diverse endophytic microorganisms. This was confirmed by light microscopy of tissue sap revealing viable bacteria in considerable numbers, which were detected under phase contrast or with negative staining. Planting tissue segments or applying homogenate from these stocks on diverse bacteriological media did not induce the organisms to grow in vitro. The shoot cultures displayed variation in growth and rooting potential, the onus of such variation was solely attributable to the associated microorganisms. The findings were confirmed with additional field shoots and fresh in vitro stocks established subsequently. The observations have implications in micropropagation and all other applications involving plant cell, tissue, organ, and protoplast culture.     

药用植物青蒿不同种类的内生菌抑菌活性分析

为了研究青蒿不同种类的内生菌抑制细菌和抑制真菌的活性,该研究采用组织块法和研磨法从青蒿的根、茎、叶中分离内生细菌、放线菌和真菌,以大肠埃希菌(Escherichia coli)(CICC 23657)、枯草芽孢杆菌(Bacillus subtilis)(CICC 10275)、金黄色葡萄球菌(Staphylococcu...     

Analysis of the Composition and Characteristics of Culturable Endophytic Bacteria Within Subnival Plants of the Tianshan Mountains, Northwestern China

This study first described the composition and characteristics of culturable endophytic bacteria isolated from wild alpine-subnival plant species growing under extreme environmental conditions (i.e., on the border of a glacier with frequently fluctuating and freezing temperatures, strong wind, and high ultraviolet radiation). Using a cultivation-dependent approach and 16S rRNA gene amplification techniques, 93 bacterial isolates showing different phenotypic properties were obtained from 20 different subnival plant species, of which gram-positive bacteria (61.5%), psychrotolerant bacteria (67.3%), and pigmented isolates (70.9%) accounted for a large proportion. All these characteristics of endophytes were closely related to the survival environment of their host plants and were in good agreement with microbes occurring in other cold environments. Phylogenetic analysis indicated that the endophytic isolates consisted of five phylogenetic groups comprising α-proteobacteria, γ-proteobacteria, the high G+C content gram-positive bacteria, the low G+C content gram-positive bacteria, and Flavobacterium-Bacteroides-Cytophaga. The largest generic diversity was found in the HGC group, while Clavibacter, Agreia, Rhodococcus, Sphingomonas, and Pseudomonas were the most prevalent genera. Of all isolates, 46.4% showed a high sequence similarity (98-100%) to strains discovered from other cold environments such as glaciers, tundra, and polar seas. Furthermore, 36.4% of the isolates produced Indole-3-acetic acid and 76.3% were able to solubilize mineral phosphate, which revealed that endophytic bacteria with multiple physiological functions were abundant and widespread in subnival plants. These results are essential for understanding the ecological roles of endophytes and as a foundation for further studying the interactions with plants and environment.     

Analysis of the bacterial community in glassy-winged sharpshooter heads

The glassy‐winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of various strains of Xylella fastidiosa, which cause disease in a variety of economically important plants. These diseases include citrus variegated chlorosis, oleander leaf scorch and Pierce's Disease of grapevines. Symbiotic control (SC) is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace the pathogenic strains of X. fastidiosa. Candidate endophytes for use in SC must occupy the xylem of host plants and attach to the pre‐cibarium and cibarium of sharpshooter insects in order to have access to the pathogen. The study of the bacterial community of GWSS heads by isolation and denaturing gradient gel electrophoresis (DGGE) revealed the presence of species that may be suitable for use in SC. In addition, the results indicated that two important factors, insect age and choice of host plant, affect the composition of the bacterial community in GWSS heads. The main bacterial genera isolated as colonizers of GWSS heads were identified, using partial 16S rRNA gene sequencing, as Bacillus, Pseudomonas, Pedobacter and Methylobacterium, as well as the species Curtobacterium flaccumfaciens. DGGE patterns revealed a diversity of endophytic species able to colonize the GWSS head. The main genera isolated in culture were also identified using this technique. Principal component analysis (PCA) from polymerase chain reaction (PCR)‐DGGE patterns indicated that the bacteria inhabiting the GWSS head are similar to those found as endophytes inside the host plants, and that insect developmental stage and preferential feeding on one host plant species over another are important factors in determining the composition of the bacterial community in the GWSS head. However, a shift in host plants for a small period of time did not cause changes in the compositions of these communities.