Endophytic Colonization of Balloon Flower by Antifungal Strain Bacillus sp. CY22

Endophytic Bacillus sp. CY22 was previously isolated from the root interior of the balloon flower (Platycodon grandiflorum) (Cho et al., Biosci. Biotechnol. Biochem., 66, 1270-1275 (2002)). Three-month-old balloon flower seedlings were inoculated with 10⁷ cfu/ml of strain CY22R3, a rifampicin-resistant strain of CY22, and external and internal root colonization was assessed 2 and 4 weeks later. After inoculation, large numbers of bacteria were observed on the root surface by scanning electron microscopy. More detailed studies using optical and transmission electron microscopy confirmed that Bacillus sp. CY22 was endophytically established within intercellular spaces, cortical cells, and aerenchymas of root. Also, Bacillus sp. CY22 showed antibiotic activities against several phytopathogens by producing the antibiotic iturin A. In the pot test, root rot of balloon flower seedlings caused by Rhizoctonia solani was suppressed when the Bacillus sp. CY22R3 was inoculated into the soil.     

Endophytic bacillus sp. isolated from the interior of balloon flower root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C15:0 (37%), C17:0 (5.1%), and iso-C15:0 (27.7%). DNA G+C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

Endophytic Bacillus sp. Isolated from the Interior of Balloon Flower Root

A bacterial strain, designated CY22, was isolated from the interior of balloon flower (Platycodon grandiflorum) root in the Republic of Korea. The isolate coproduced an iturin-like antifungal compound and a surfactin-like potent biosurfactant. Analysis of the 16S-rDNA of strain CY22 showed that the isolate was a member of Bacillus. High similarities were observed between strain CY22 and Bacillus sp. TKSP 24, and between strain CY22 and B. subtilis 168. Phylogenetic analysis based on 16S-rDNA sequences showed that strain CY22 was closely related to Bacillus sp. The main whole-cell fatty acids were anteiso-C_15:0 (37%), C_17:0 (5.1%), and iso-C_15:0 (27.7%). DNA G + C content was 54 mol%. Based on phylogenetic inference, phenotypic and chemotaxonomic characteristics, this endophytic strain Bacillus sp. CY22 was assigned to the genus Bacillus.     

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.     

Endophytic colonization ability of two deep-water rice endophytes, Pantoea sp. and Ochrobactrum sp. using green fluorescent protein reporter

Colonization ability of the two endophytic bacteria, isolated from surface sterilized seeds of Jaisurya variety of deep-water rice viz., Pantoea sp. and Ochrobactrum sp., was compared after genetically tagging them with a constitutively expressing green fluorescent protein gene (gfp). Confocal laser scanning microscopy (CLSM) of hydroponically grown seedlings of Jaisurya rice, inoculated with gfp-tagged endophytes, revealed that both Pantoea sp. and Ochrobactrum sp. colonized the intercellular spaces in the root cortex when inoculated separately. Colonization by gfp-tagged Ochrobactrum sp. was severely inhibited when co-inoculated with an equal number (10(5) c.f.u. ml(-1)) of wild type Pantoea sp., but the converse was not true. Pantoea sp. was a more aggressive endophytic colonizer of its host than Ochrobactrum sp. The potential of using GFP reporter and CLSM as tools in evaluating competitive ability of colonization among endophytes is herewith demonstrated.     

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.     

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).     

Isolation and characterization of Bacillus sp. producing broad-spectrum antibiotics against human and plant pathogenic fungi

A strain of bacterium producing antifungal antibiotic was isolated and identification of the strain was attempted. We could identify the bacterium as being a Bacillus sp., based on morphological observation, physiological characteristics, and 16S rDNA sequence analysis, thus leading us to designate the strain as Bacillus sp. AH-E-1. The strain showed potent antibiotic activity against phytopathogenic and human pathogenic fungi by inducing mycelial distortion and swelling and inhibiting spore germination. The antibiotic metabolite produced by the strain demonstrated excellent thermal and pH (2-11) stability, but was labile to autoclaving. From these results, we could find a broader antifungal activity of Bacillus genus. Isolation and characterization of the active agent produced by the strain are under progress.     

Effects of nematodes,fungi and bacteria on the growth of young apple trees grown in apple replant disease soil

Growth, dry root weight of seedlings and root score of apple seedlings cv. McIntosh were reduced when soils were inoculated with Pratylenchus penetrans, Penicillium janthinellum, Constantinella terrestris, Trichoderma sp., and 4 strains of Bacillus subtilis. Trichoderma sp., and B-1 and B-26 strains of B. subtilis alone reduced plant growth but the combination of Trichoderma sp. + B. subtilis (B-1) and Trichoderma sp. + B. subtilis (B-26) increased plant height. Plant height, root weight and root score were significantly reduced when P. penetrans plus B. subtilis or P. penetrans plus fungi plus bacteria were present in the soil. It is suggested that fungi, bacteria, nematodes alone or their combinations such as nematodes plus bacteria or nematodes plus fungi plus bacteria may contribute towards the occurrence of apple replant disease.Contribution number 700.Contribution number 700.     

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43)

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.     

Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglasfir seedlings

The survival, development and mycorrhizal efficiency of a selected strain of Laccaria bicolor along with naturally occurring ectomycorrhizal fungi in a young plantation of Douglas fir was examined. Symbionts were identified and their respective colonization abilities were determined. Eight species of symbiotic fungi, which may have originated in adjacent coniferous forests, were observed on the root systems. Mycorrhizal diversity differed between inoculated (5 taxa) and control (8 taxa) seedlings. Ectomycorrhizal fungi which occurred naturally in the nursery on control seedlings (Thelephora terrestris and Suillus sp.) did not survive after outplanting. Both inoculated and naturally occurring Laccaria species, as well as Cenococcum geophilum, survived on the old roots and colonized the newly formed roots, limiting the colonization by other naturally occurring fungi. Other fungi, such as Paxillus involutus, Scleroderma citrinum and Hebeloma sp. preferentially colonized the old roots near the seedling's collar. Russulaceae were found mainly in the middle section of the root system. Mycorrhizal colonization by Laccaria species on inoculated seedlings (54%) was significantly greater than on controls (13%) which were consequently dominated by the native fungi. Significant differences (up to 239%) were found in the growth of inoculated seedlings, especially in root and shoot weight, which developed mainly during the second year after outplanting. Seedling growth varied with the species of mycorrhizae and with the degree of root colonization. Competitiveness and effectiveness of the introduced strain on improving growth performances of seedlings are discussed.     

卡特兰与丝核菌共培养体系的建立及卡特兰菌根显微结构的研究*

用3个分离自野生卡特兰的丝核菌菌株接种卡特兰组培幼苗,对幼苗生长有不同程度的促进作用,处理苗的鲜重增长率(%)分别是67.5、67.3和62.4,而不接菌的对照仅为49.5,其中KW214菌株处理苗与对照差异达显著水平(α=0.05)。接菌处理后植株的N、P、K含量没有明显增加。在三个接菌处理苗的营养根中均分离获得了原接种真菌,并观察了接种KW214菌株的卡特兰营养根。真菌先侵入根被组织,经通道细胞最后侵染皮层组织细胞,并通过菌丝穿越细胞壁不断地向内延伸扩展。菌丝在皮层组织细胞内形成大量着色较深形状不规则的菌丝结等兰科菌根典型结构,菌丝结在皮层组织中分布不均,多出现在靠外侧的几层皮层细胞内,在被侵染的细胞中,菌丝结常出现在细胞核附近。菌丝结、针状晶体和细胞核在形态和着色反应上有明显区别。菌丝结和细胞核常常相互靠近。从生物量增长、显微结构、重分离接种菌株等三个方面获得的证据表明,已成功建立了卡特兰组培幼苗和丝核菌KW214的共培养体系。     

Recognition of Leguminous Hosts by a Promiscuous Rhizobium Strain

The lima bean (Phaseolus lunatus L.) and the pole bean (Phaseolus vulgaris L.) are nodulated by rhizobia of two different cross-inoculation groups. Rhizobium sp. 127E15, a cowpea-type Rhizobium, can induce effective nodules on the lima bean and partially effective nodules on the pole bean. Rhizobium phaseoli 127K14 can induce effective nodules on the pole bean but does not reciprocally nodulate the lima bean. Root hairs of the lima bean when inoculated with Rhizobium sp. 127E15 showed tip curling and swelling and infection thread formation as observed by light microscopy and scanning electron microscopy. When lima bean root hairs were inoculated with R. phaseoli 127K14, no host-specific responses were observed. Pole bean root hairs that had been inoculated with R. phaseoli 127K14 or Rhizobium sp. 127E15 also showed tip curling and swelling and infection thread formation. Colonization of lima bean root hairs by Rhizobium sp. 127E15 and pole bean root hairs by R. phaseoli 127K14 or Rhizobium sp. 127E15 appeared to involve the elaboration of microfibrils. This study showed that when Rhizobium sp. 127E15 nodulates a host of a different cross-inoculation group, it elicits the same specific host responses as it does from a host of the same cross-inoculation group.     

金荞麦和苦荞麦抗菌活性内生真菌的筛选及鉴定

从药用植物金荞麦和苦荞麦的根、茎、叶、花中分离到62株内生真菌,并以金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)[CMCC(B)63501]、小麦赤霉病菌(Fusarium graminearum)、黄瓜枯萎病菌(Fusarium oxysporum f.sp.cu-cumerinum)和绵腐病菌(Pythium aphanidermatum)6种微生物为指示菌对分离到的内生真菌进行抗菌活性检测。结果发现,分离的内生真菌菌株KQH-01、KQH-02和JQY-1的发酵醇提取物具有较好的抑菌活性。形态学特征和分子鉴定确定菌株KQH-01为炭角菌属(Xylaria sp.)真菌,菌株KQH-02为球毛壳菌(Chaetomium globosum),菌株JQY-1为葡萄座腔菌(Botryosphaeria dothidea)。     

Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth

Some pseudomonads produce a toxin that specifically inhibits winter wheat (Triticum aestivum L.) root growth and the growth of several microorganisms. The toxin does not inhibit pea (Pisum sativum) root growth, but the organisms are aggressive root colonizers and their effect on Rhizobium leguminosarum growth, colonization, and nodulation of peas was not known. Peas were grown in Leonard jars in the greenhouse. Pea roots were inoculated with R. leguminosarum, a toxin-producing Pseudomonas sp., both, or neither (control). The Pseudomonas sp. colonized pea roots more rapidly and in greater number than R. leguminosarum after ten days. In the presence of the Pseudomonas sp., the R. leguminosarum population on the rhizoplane was less at ten days. When the roots were inoculated with both R. leguminosarum and Pseudomonas sp., the number of nodules were greater than when R. leguminosarum was inoculated alone, but nodule dry weight and pea shoot biomass were similar to plants inoculated with only R. leguminosarum. Although these results need confirmation with non-sterile soil and field studies, these preliminary results indicate that peas will not be affected by wheat root-inhibitory rhizobacteria.     

Co-inoculation with Bacillus sp. CECT 450 improves nodulation in Phaseolus vulgaris L.

The strain Bacillus sp. CECT 450 increased nodulation on bean (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici CIAT 899. This positive effect occured under controlled conditions on perlite-vermiculite, sand, or in a mixture of soil and sand. This increase was also observed in a field assay. Nodulation kinetic studies suggested that the synergistic effect is pronounced during the latter stages of cultivation. In contrast, the same bacteria co-inoculated with Bradyrhizobium japonicum USDA 110 reduced nodulation on soybean (Glycine max (L.) Merr.). Inoculation with Bacillus sp. CECT 450 alone had no effect on bean plants, but reduced root growth in soybean. The survival of Bacillus sp. CECT 450 on inoculated seeds was high, even when inoculated seeds were maintained for several months at room temperature.     

Stimulation of the ionic transport system in Brassica napus by a plant growth-promoting rhizobacterium (Achromobacter sp.)

A plant growth-promoting rhizobacterium belonging to the genus Achromobacter was isolated from the oil-seed-rape (Brassica napus) root. Growth promotion bioassays were performed with oilseed rape seedlings in a growth chamber in test tubes containing attapulgite and mineral nutrient solution, containing NO3- as N source. The presence of this Achromobacter strain increased shoot and root dry weight by 22-33% and 6-21%, respectively. Inoculation of young seedlings with the Achromobacter bacteria induced a 100% improvement in NO3- uptake by the whole root system. Observations on the seminal root of seedlings 20 h after inoculation showed that there was an enhancement of both the number and the length of root hairs, compared to non-inoculated seedlings. Electrophysiological measurements of NO3- net flux with ion-selective microelectrodes showed that inoculation resulted in a specific increase of net nitrate flux in a root zone morphologically similar in inoculated and non-inoculated plants. The root area increased due to root hair stimulation by the Achromobacter bacteria, which might have contributed to the improvement of NO3- uptake by the whole root system, together with the enhancement of specific NO3- uptake rate. Moreover, inoculated plants showed increased potassium net influx and proton net efflux. Overall, the data presented suggest that the inoculation of oilseed-rape with the bacteria Achromobacter affects the mineral uptake.     

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...     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

外生菌根菌对樟子松苗木生长的影响

通过菌种野外采集、分离培养、纯化及室内人工接种试验,获得9株樟子松外生菌根菌。采用苗木截根—菌剂浸根方法,对2年生樟子松移床苗进行外生菌根菌野外单接种试验,以接种无菌液体培养基的苗木作为对照,研究外生菌根菌单接种对樟子松苗木生长的影响。野外单接种试验表明,9株外生菌根菌对樟子松苗木均有一定的促生长效果。接种130 d,除菌株010外,其他菌株均可提高樟子松苗高23%以上。其中,接种菌株GT005的苗高提高54.24%;接种菌株035、009、LH004及GT001的苗高分别提高41.53%、36.44%、35.59%和35.59%。菌株035、LH004、025、010和GT001可使苗木地径提高20%以上,其中菌株035提高地径56.31%,菌株LH0040、25分别提高39.93%和29.01%。除接种菌株010的苗木外,接种其他菌株的樟子松苗木过氧化氢酶活性均高于对照30.23%~48.37%。接种菌株004的苗木根系活力最强,高于对照39.17%;接种菌株GT005和010的苗木,根系活力低于对照;接种其他菌株的苗木根系活力高于对照2.5%~16.67%。接种菌株GT005的苗木的叶绿素含量最高,高于对照20%以上。9个菌株均可用于樟子松苗木生产中。樟子松苗木的过氧化氢酶活性、根系活力与苗木的生物量间无相关性。