砂生槐根瘤内生菌对青稞种子萌发及幼苗的促生作用

该文以接种不同砂生槐根瘤内生菌的黑青稞种子和不接种菌株的黑青稞种子为研究材料,采用培养皿作为基质,观察黑青稞种子萌发及幼苗生长情况,测定接种不同根瘤内生菌后的黑青稞发芽率、发芽势、发芽指数、株高、根长、须根数、根冠比、植株全株干/鲜重的生理性状,并对各不同处理进行隶属函数综合评价.结果表明:菌株R7和R17的发芽率均达...     

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.     

Model plants for studying the interaction between Methylobacterium mesophilicum and Xylella fastidiosa

Over the last few years, endophytic bacterial communities associated with citrus have been studied as key components interacting with Xylella fastidiosa. In this study, we investigated the possible interaction between the citrus endophyte Methylobacterium mesophilicum SR1.6/6 and X. fastidiosa in model plants such as Catharanthus roseus (Madagaskar periwinkle) and Nicotiana clevelandii (Clevelands tobacco). The aim of this study was to establish the fate of M. mesophilicum SR1.6/6 after inoculation of C. roseus and N. clevelandii plants, using PCR-DGGE (polymerase chain reaction--denaturing gradient gel electrophoresis) and plating techniques. Shifts in the indigenous endophytic bacterial communities were observed in plants inoculated with strain SR1.6/6, using specific primers targeting alpha- and beta-Proteobacteria. Cells of strain SR1.6/6 were observed in a biofilm structure on the root and hypocotyl surfaces of in vitro seedlings inoculated with M. mesophilicum SR1.6/6. This emphasizes the importance of these tissues as main points of entrance for this organism. The results showed that C. roseus and N. clevelandii could be used as model plants to study the interaction between M. mesophilicum and X. fastidiosa.     

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.     

Effect of bacterial antagonists on lettuce: active biocontrol of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms

The aim of this study was to assess the biocontrol efficacy against Rhizoctonia solani of three bacterial antagonists introduced into naturally Rhizoctonia-infested lettuce fields and to analyse their impact on the indigenous plant-associated bacteria and fungi. Lettuce seedlings were inoculated with bacterial suspensions of two endophytic strains, Serratia plymuthica 3Re4-18 and Pseudomonas trivialis 3Re2-7, and with the rhizobacterium Pseudomonas fluorescens L13-6-12 7 days before and 5 days after planting in the field. Similar statistically significant biocontrol effects were observed for all applied bacterial antagonists compared with the uninoculated control. Single-strand conformation polymorphism analysis of 16S rRNA gene or ITS1 fragments revealed a highly diverse rhizosphere and a less diverse endophytic microbial community for lettuce. Representatives of several bacterial (Alpha-, Beta- and Gammaproteobacteria, Firmicutes, Bacteriodetes), fungal (Ascomycetes, Basidiomycetes) and protist (Oomycetes) groups were present inside or on lettuce plants. Surprisingly, given that lettuce is a vegetable that is eaten raw, species of genera such as Flavobacterium, Burkholderia, Staphylococcus, Cladosporium and Aspergillus, which contain potentially human pathogenic strains, were identified. Analysis of the indigenous bacterial and endophytic fungal populations revealed only negligible, short-term effects resulting from the bacterial treatments, and that they were more influenced by field site, plant growth stage and microenvironment.     

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.     

Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens

Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var. Shinano No. 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121). The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants). The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively. Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A. tumefaciens harboring a modified pBI121 for plasmid rescue. Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.     

Characterization and screening of plant probiotic traits of bacteria isolated from rice seeds cultivated in Argentina

Many seeds carry endophytes, which ensure good chances of seedling colonization. In this work, we have studied the seed-borne bacterial flora of rice varieties cultivated in the northeast of Argentina. Surface-sterilized husked seeds of the rice cultivars CT6919, El Paso 144, CAMBA, and IRGA 417 contained an average of 5×10⁶ CFU/g of mesophilic and copiotrophic bacteria. Microbiological, physiological, and molecular characterization of a set of 39 fast-growing isolates from the CT6919 seeds revealed an important diversity of seed-borne mesophiles and potential plant probiotic activities, including diazotrophy and antagonism of fungal pathogens. In fact, the seed-borne bacterial flora protected the rice seedlings against Curvularia sp. infection. The root colonization pattern of 2 Pantoea isolates from the seeds was studied by fluorescence microscopy of the inoculated axenic rice seedlings. Both isolates strongly colonized the site of emergence of the lateral roots and lenticels, which may represent the entry sites for endophytic spreading. These findings suggest that rice plants allow grain colonization by bacterial species that may act as natural biofertilizers and bioprotectives early from seed germination.     

Dynamics of seed-borne rice endophytes on early plant growth stages

Bacterial endophytes are ubiquitous to virtually all terrestrial plants. With the increasing appreciation of studies that unravel the mutualistic interactions between plant and microbes, we increasingly value the beneficial functions of endophytes that improve plant growth and development. However, still little is known on the source of established endophytes as well as on how plants select specific microbial communities to establish associations. Here, we used cultivation-dependent and -independent approaches to assess the endophytic bacterrial community of surface-sterilized rice seeds, encompassing two consecutive rice generations. We isolated members of nine bacterial genera. In particular, organisms affiliated with Stenotrophomonas maltophilia and Ochrobactrum spp. were isolated from both seed generations. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) of seed-extracted DNA revealed that approximately 45% of the bacterial community from the first seed generation was found in the second generation as well. In addition, we set up a greenhouse experiment to investigate abiotic and biotic factors influencing the endophytic bacterial community structure. PCR-DGGE profiles performed with DNA extracted from different plant parts showed that soil type is a major effector of the bacterial endophytes. Rice plants cultivated in neutral-pH soil favoured the growth of seed-borne Pseudomonas oryzihabitans and Rhizobium radiobacter, whereas Enterobacter-like and Dyella ginsengisoli were dominant in plants cultivated in low-pH soil. The seed-borne Stenotrophomonas maltophilia was the only conspicuous bacterial endophyte found in plants cultivated in both soils. Several members of the endophytic community originating from seeds were observed in the rhizosphere and surrounding soils. Their impact on the soil community is further discussed.     

Colonization behavior of bacterium Burkholderia cepacia inside the Oryza sativa roots visualized using green fluorescent protein reporter

Colonization behavior of endophytic bacteria Burkholderia cepacia strains RRE-3 and RRE-5 was studied in the seedlings of rice variety NDR97 using confocal laser scanning microscopy under controlled laboratory and greenhouse conditions. For studying colonization pattern, bacterial strains were tagged with pHRGFPGUS plasmid. The role of bacterial strains (both gfp/gus-tagged and untagged) in growth promotion was also studied. After coming into contact with the host root system the bacteria showed an irregular spreading. Dense colonization was observed on the primary and secondary roots and also on the junction of emergence of the lateral roots. Results showed that the colonization pattern of Burkholderia cepacia strains was similar to that of other endophytic bacteria isolated from non-legumes. Burkholderia cepacia got entry inside the root at the sites of emergence of lateral roots, without formation of infection threads as in the case of symbiotic rhizobacteria. Observations suggested that the endophytic bacterial strains RRE-3 and RRE-5 entered inside the rice roots in a progressive manner. Bacteria were found to line up along the intercellular spaces of adjoining epidermal cells adjacent to the lateral root junction, indicating endophytic colonization pattern of Burkholderia cepacia strains. Experiments with the rice seedlings inoculated with RRE-3 and RRE-5 strains revealed that both strains enhanced plant growth considerably when observed under laboratory and greenhouse conditions and produced significantly higher plant biomass. No considerable difference was observed between the gfp/gus-tagged and non-gfp/gus-tagged strains in the plant growth experiments both in the laboratory and greenhouse conditions.     

Germination of the seeds of ulluco (Ullucus tuberosus,Basellaceae)

Seeds of eight clones of ulluco (Ullucus tuberosus) were collected in 1985–1986 in cultivation experiments in the Botanical Garden of the University of Turku, and their germination was studied in a greenhouse and a growth chamber. The number of tested seeds was 1177. The disinfected seeds were tested in 10 different treatments with different illumination rhythms, temperatures, duration of treatments, and media. Stratification, removal of the calyx and seed coat, and mechanical rupture of the seed coat were used in the treatments. The mean germination percentage was 2.04%, but only five seedlings were obtained. Germinated seeds were obtained in three of the treatments; they belonged to clones from Puno, Cusco, and Juliaca. The best result was obtained by treating the seeds with 0.1% gibberellic acid, but the best seedlings, from the Puno clone, developed in pure water. The shortest germination time was 60 d and the longest 650 d, even within the same clone. The best illumination for germination was 8 h light and 16 h dark, in temperatures of +20°C (day) and +6°C (night). The seeds were found to germinate even after a storage period of 18 mo. The seedlings varied in habit, in size of seedling leaves, and in colour of stem, which could be light green or reddish.     

Application of endophytic bacteria for the biocontrol of Rhizoctonia solani (Cantharellales: ceratobasidiaceae) damping-off disease in cotton seedlings

The antifungal potentialities of three endophytic bacterial strains, Stenotrophomonas maltophila H8 (Xanthomonadales: Xanthomonadaceae), Pseudomonas aeruginosa H40 (Pseudomonadales: Pseudomonadaceae) and Bacillus subtilis H18 (Bacillales: Bacillaceae) were evaluated against the phytopathogenic fungus Rhizoctonia solani in cotton seedlings under greenhouse conditions. The bacterial strains were applied as a soil drench or talc-based bioformulation in R. solani-infested soil and non-infested soil. Results indicated that the soil drench treatment was more efficient than talc-based bioformulation. A significant increase of seed emergence and seedling survival with a clear reduction of disease severity was achieved with the endophytic bacterial treatments. At the same time, the fresh weight, dry weight, shoot length and root length of the treated plants were markedly enhanced. Moreover, there was an apparent induction of the antioxidant enzymes (peroxidase, polyphenol oxidase and catalase) of the treated seedlings. Gas chromatography–mass spectrometry revealed the presence of various bioactive compounds in the bacterial supernatant. The antagonistic activity of the bacterial strains against R. solani was attributed to their capability to produce a broad spectrum of antifungal compounds in addition to bioactive molecules that can trigger the systemic resistance in the infected seedlings.     

Crude oligosaccharides mediated resistance and histo-chemical changes in Capsicum annuum against anthracnose disease caused by Colletotrichum capsici

Enhancing the host resistance using biotic elicitors is one of the eco-friendly approaches developed for plant disease management. The Crude Oligosaccharides (CO) extracted from Colletotrichum capsici (Syd.) Butler & Bisby was evaluated for their efficiency to elicit resistance in chilli against anthracnose disease. Among the different concentrations tested, CO treatment at 2.5 mg/ml concentration for 3 h duration significantly enhanced seed germination (90.5%) and seedling vigor (986.7), compared to control which offered 78% of seed germination and 712.5 of seedling vigor. Application of CO at 2.5 mg/ml concentration also reduced the disease severity with a highest anthracnose disease protection of 68% under greenhouse conditions and enhanced the vegetative growth parameters compared to control. The induction of resistance was evident with higher expression of primary defense responses like hypersensitive response, deposition of lignin, callose, hydrogen peroxide and phenol when compared to control plants. There was a one fold increase in defense enzyme activities phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, and lipoxygenase in crude oligosaccharide-treated inoculated seedlings when compared to susceptible inoculated seedlings which were similar to that of resistant inoculated seedlings where a maximum of 1.5-fold increase in enzyme activity was observed.     

How significant are endophytic fungi in bromeliad seeds and seedlings? Effects on germination,survival and performance of two epiphytic plant species

In bromeliads, nothing is known about the associations fungi form with seeds and seedling roots. We investigated whether fungal associations occur in the seeds and seedling roots of two epiphytic Aechmea species, and we explored whether substrate and fungal associations contribute to seed germination, and seedling survival and performance after the first month of growth. We found a total of 21 genera and 77 species of endophytic fungi in the seeds and seedlings for both Aechmea species by Illumina MiSeq sequencing. The fungal associations in seeds were found in the majority of corresponding seedlings, suggesting that fungi are transmitted vertically. Substrate quality modulated the germination and growth of seedlings, and beneficial endophytic fungi were not particularly crucial for germination but contributed positively to survival and growth. Overall, this study provides the first evidence of an endophytic fungal community in both the seeds and seedlings of two epiphytic bromeliads species that subsequently benefit plant growth.     

Endophytic bacteria of the rock-dwelling cactus Mammillaria fraileana affect plant growth and mobilization of elements from rocks

Mammillaria fraileana is a major pioneer, small cactus that harbors endophytic bacteria that have plant growth-promoting traits, including rock-weathering capacity. Our working hypothesis was that this functional group of endophytic bacteria assists in establishing pioneer plants on rocks. When these endophytic bacteria were inoculated on seedlings grown in rock substrate, mobilization of elements from the substrate increased at variable levels across combinations of substrates and inoculants. In plants grown in the rhyodacite substrate, where these cacti naturally grow, increased mobilization occurred in plants inoculated with several strains. Promotion of plant growth, manifested as an increase in dry weight, was greater in cacti inoculated with Enterobacter sakazakii M2PFe. Accumulation of nocturnal acids, indicating photosynthesis by crassulacean acid metabolism, was superior in plants inoculated with the endophytes Azotobacter vinelandii M2Per and Pseudomonas putida M5TSA. Inoculation with endophytes can stimulate plant growth of M. fraileana by mobilizing elements from rock, which can lead to higher photosynthetic activity and accumulation of biomass. Inoculation with P. putida M5TSA also led to accumulation of more total nitrogen than plants inoculated with a control nitrogen-fixing bacteria. Evidence of endophytic colonization is provided after initial inoculation of seedlings and re-isolation and sequencing of 16S DNA of recovered bacteria from developing disinfected plants. The associative interaction between pioneer cacti and their bacterial endophytes enable the host plants to grow in places where plants do not normally grow. Through colonization and establishment of pioneer plants, soil is created, which facilitates colonization by other desert species and contributes to the diversity of dry lands.     

Selection of Bacillus thuringiensis strains in citrus and their pathogenicity to Diaphorina citri (Hemiptera: Liviidae) nymphs

Bacillus thuringiensis (Bt) toxins are effective in controlling insect pests either through the spraying of products or when expressed in transgenic crops. The discovery of endophytic Bt strains opened new perspectives for studies aimed at the control of sap‐sucking insects, such as the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae), a vector of “Candidatus Liberibacter spp.,” associated with citrus huanglongbing (HLB). In this study, translocation of endophytic Bt strains in citrus seedlings inoculated with Bt suspension delivered by soil‐drench, and their systemic pathogenicity to D. citri nymphs were investigated. The pathogenicity of three wild‐type Bt strains against D. citri third‐instar nymphs was demonstrated. Among the 10 recombinant strains tested (each of them harboring a single cry or cyt gene), 3 can be highlighted, causing 42%–77% and 66%–90% nymphal mortality at 2 and 5 d after inoculation, respectively. The isolation of Bt cells from young citrus shoots and dead nymphs, and PCR performed with specific primers, confirmed the involvement of the Bt strains in the psyllid mortality. This is the first report showing the translocation of Bt strains from citrus seedling roots to shoots and their potential to control D. citri nymphs that fed on these soil‐drench inoculated seedlings. The Bt strains that caused the highest mortality rates have the potential to be used as bioinsecticides to control D. citri and the identified genes can be used for the production of transgenic Bt citrus.     

Chromate resistantBacillus cereus augments sunflower growth by reducing toxicity of Cr (VI)

Impact of four chromium resistant bacterial strains (S3, S4, S6, and S7) was studied on the different growth parameters of sunflower (Helianthus annuus var SF-187) in chromium free or under chromium stress. Strains used exhibited very high-level resistance to chromate (up to 50 mg ml^-1 on nutrient agar and 1-2 mg ml^-1 in minimal medium). Application of Cr(VI) salt adversely affected the seed germination, root and shoot length, and fresh weight of seedlings. Bacterial inoculations improved the growth parameters. The effects of Cr(VI) on the different biochemical parameters were also very severe but seedlings inoculated with bacteria showed much improvements as compared to non-inoculated controls. Uptake of Cr(VI) was higher than Cr(III) by the seedlings. Inoculated seedlings contained less chromium than non-inoculated seedlings. Much improvement in the internal region of root and shoot was observed in inoculated plants especially in guard cells.     

Endophytic bacteria isolated from orchid and their potential to promote plant growth

Twelve endophytic bacteria were isolated from the meristem of in vitro Cymbidium eburneum orchid, and screened according to indole yield quantified by colorimetric assay, in vitro phosphate solubilization, and potential for plant growth promotion under greenhouse conditions. Eight strains with positive results were classified into the genus Paenibacillus by FAME profile, and evaluated for their ability to increase survival and promote the growth of in vitro germinated Cattleya loddigesii seedlings during the acclimatization process. The obtained results showed that all strains produced detectable indole levels and did not exhibit potential for solubilizing inorganic phosphate. Particularly, an increase of the total biomass and number of leaves was observed. Two strains of Paenibacillus macerans promoted plant growth under greenhouse conditions. None of the treatments had a deleterious effect on growth of inoculated plants. These results suggest that these bacterial effects could be potentially useful to promote plant growth during seedling acclimatization in orchid species other than the species of origin.     

豚草种带内生真菌及其对种子发芽和幼苗生长的影响

内生真菌是一类共生于植物体内,能够不同程度影响宿主植物生态适应性和竞争能力的微生物。分析内生真菌在豚草种子中的分布、种群结构,以及内生真菌发酵液对种子发芽和幼苗生长的作用。结果显示:发生于6个地区的豚草种子均能分离获得内生真菌,分离率在19%—92.63%之间,不同地区之间差异极显著(P0.01)。内生真菌主要存在于种子的总苞部位,分离率达到65.52%。发生于福建省长乐市松下镇的豚草种带内生真菌种群包含5个属,以链格孢属(Alternaria)真菌为优势菌群,占82.26%;其次为镰孢属(Fusarium)真菌,占9.68%;其它3个属的真菌发生较少,均低于5%。内生真菌主要以水平传播方式在豚草不同世代之间传播。供试的7个内生真菌菌株的发酵液均不同程度地抑制豚草种子发芽,降低幼苗地上部高度、根长度、根数量和总生物量,但不同菌株发酵液之间抑制程度差异明显,显示不同菌株对豚草种子发芽和幼苗生长产生不同的影响。内生真菌发酵液处理后的种子仍然保持较高程度的活力;不同内生真菌发酵液处理后,有活力的种子维持在50%—87.5%之间,均高于(或等于)清水处理的种子,说明内生真菌代谢产物只是抑制种子的发芽,但并不导致种子的腐烂和死亡。这些研究结果初步显示种子携带的内生真菌可能在豚草入侵生物学中发挥重要的作用。