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.     

具ACC脱氨酶活性的碱蓬内生细菌的分离、鉴定及其生物学特性

【目的】具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的盐生植物碱蓬内生细菌的分离及生物学特性的研究有助于探索内生细菌与宿主植物耐盐性的关系。【方法】采用研磨法从健康碱蓬植株的根、茎、叶中分离具有ACC脱氨酶活性的碱蓬内生细菌,根据形态特征、生理生化、API鉴定系统和16S rRNA对菌种进行鉴定,并分别测定了菌株产ACC脱氨酶、铁载体、吲哚乙酸、赤霉素、脱落酸、蛋白酶及溶磷、固氮和拮抗病原菌的特性。【结果】将分离得到的内生细菌LP11、SS12、TW1和TW2分别鉴定为栖稻假单胞菌(Pseudomonas oryzihabitans)、假单胞菌(Pseudomonas sp.)、成团泛菌(Pantoea agglomerans)和恶臭假单胞菌(Pseudomonas putida),4株菌除具有较高的ACC脱氨酶活力之外,还可不同程度地产生铁载体、吲哚乙酸、赤霉素和脱落酸,且均有溶磷作用,但无固氮能力及蛋白酶活力,唯有菌株SS12对萝卜枯萎病菌(Fusariumoxysporum f.sp.conglutinans)和黄瓜枯萎病菌(F.oxysporum f.sp.cucumerinum)具有拮抗作用。【结论】从盐生植物碱蓬中分离到的假单胞菌属和泛菌属内生细菌,具有丰富多样的生物学特性。     

Isolation and identification of marine chitinolytic bacteria and their potential in antifungal biocontrol

Chitinolytic marine bacterial strains (30) were isolated from the sea dumps at Bhavnagar, India. They were screened as chitinase producers on the basis of zone of clearance on chitin agar plates incorporated with calcofluor white M2R for the better resolution. Out of these, three strains namely, Pseudomonas sp., Pantoea dispersa and Enterobacter amnigenus showed high chitinase production. They were also found to produce proteases and therefore have a good potential for use as antifungal biocontrol agents for the control of fungal plant pathogens. These strains could degrade and utilize the mycelia of Macrophomina phaseoliena (Tassi) Goidanich and Fusarium sp. In vitro, these strains could inhibit the growth of Fusarium sp. and M. phaseolina. The culture filtrate inhibiting hyphal elongation was observed microscopically.     

Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks

Fungi and bacteria were isolated from surface disinfected leaf tissues of several citrus rootstocks. The principal bacterial species isolated were Alcaligenes sp., Bacillus spp. (including B. cereus, B. lentus, B. megaterium, B. pumilus, and B. subtilis), Burkholderia cepacia, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium extorquens, and Pantoea agglomerans, with P. agglomerans and B. pumilus being the most frequently isolated species. The most abundant fungal species were Colletotrichum gloeosporioides, Guignardia citricarpa, and Cladosporium sp. Genetic variability between 36 endophytic bacterial isolates was analysed by the random amplified polymorphic DNA (RAPD) technique, which indicated that B. pumilus isolates were more diverse than P. agglomerans isolates, although genetic diversity was not related to the host plants. In vitro interaction studies between G. citricarpa isolates and the most frequently isolated endophytic bacteria showed that metabolites secreted by G. citricarpa have an inhibitory growth effect on some Bacillus species, and a stimulatory growth effect on P. agglomerans.     

Effect of increased temperature in apical regions of maize ears on starch-synthesis enzymes and accumulation of sugars and starch

Apical florets of maize (Zea mays L.) ears differentiate later than basal florets and form kernels which have lower dry matter accumulation rates. The purpose of this study was to determine whether increasing the temperature of apical kernels during the dry matter accumulation period would alter the difference in growth rate between apical and basal kernels. Apical regions of field-grown maize (cultivar Cornell 175) ears were heated to 25 ± 3°C from 7 days after pollination to maturity (tip-heated ears) and compared with unheated ears (control). In controls, apical-kernel endosperm had 24% smaller dry weight at maturity, lower concentration of sucrose, and lower activity of ADP-Glc starch synthase than basal-kernel endosperm, whereas ADP-Glc-pyrophosphorylase (ADPG-PPase) activities were similar. In tip-heated ears apical-kernel endosperm had the same growth rate and final weight as basal-kernel endosperm and apical kernels had higher sucrose concentrations, higher ADP-Glc starch synthase activity, and similar ADPG-PPase activity. Total grain weight per ear was not increased by tip-heating because the increase in size of apical kernels was partially offset by a slight decrease in size of the basal- and middle-position kernels. Tip-heating hastened some of the developmental events in apical kernels. ADPG-PPase and ADP-Glc starch synthase activities reached peak levels and starch concentration began rising earlier in apical kernels. However, tip-heating did not shorten the period of starch accumulation in apical kernels. The results indicate that the lower growth rate and smaller size of apical kernels are not solely determined by differences in prepollination floret development.     

Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.     

Genome sequence of Pantoea sp. strain Sc 1, an opportunistic cotton pathogen

Pantoea is comprised of a broad spectrum of species, including plant pathogens. Here, we provide an annotated genome sequence of Pantoea sp. strain Sc 1, which was isolated from a diseased cotton boll. This research provides the first genome sequence of a bona fide Pantoea sp. insect-vectored cotton pathogen.     

Peanut Pod Invasion by Aspergillus flavus in the Presence of Meloidogyne hapla

''Argentine'', ''Early Runner'' and ''Florigiant'' peanut cultivars were grown in methyl bromide treated soil in field microplots inoculated with: (i) Aspergillus flavus or (ii) A. flavus + Meloidogyne hapla. Nematode infection produced heavy root galling and light pod galling equally on all cultivars. A. flavus, A. niger, Cephalosporium spp., Colletotrichum sp., Curvularia spp., Fusarium spp., Penicillium spp. and Trichoderma viride were isolated from shells and kernels. A significantly greater incidence and density of A. flavus was obtained from kernels of plants inoculated with both organisms than from kernels of plants receiving only the fungus. Differences were not significant, however, for incidence and density of A. flavus in shells or for the total of all fungal propagules in shells and kernels. Shells of ''Early Runner'' contained significantly greater incidence and density of A. flavus than the other two cultivars; also, kernels of this cultivar contained more fungal propagules than kernels of ''Argentine.'' A significantly larger number of total fungi was isolated from kernels of ''Argentine'' than from ''Florigiant.'' Aflatoxins were found only in two shell samples and not in kernels.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

杂交水稻种子固有细菌群落多样性探究

采用传统的分离培养方法和分子生物学技术对我国高产杂交水稻(OryzasativaL.)金优611种子固有细菌进行研究,从而了解其中可培养细菌群落的多样性。对分离得到的91株细菌进行16SrDNA扩增、ARDRA分型和16SrDNA系统发育分析,结果表明,分离得到的91株细菌分属于10个属16个种。其中γ-变形杆菌(Gammaproteobacteria)(53.85%)占据优势地位,其次为α-变形杆菌(Alphaproteobacteria)(20.88%),其它分属放线菌门Actinobacteria(15.39%)及厚壁菌门Firmicutes(9.88%)。其中的泛菌属(Pantoea sp.)和鞘氨醇单胞菌属(Sphingomonas sp.)、假单胞菌属(Pseudomonas sp.)、微杆菌属(Microbacterium sp.)为分离到的优势种群,且在种子这一特殊的生存空间中有4株潜在的新种存在。首次报道了杂交水稻金优611种子具有丰富的微生物群落多样性,为进一步探索植物种子际微生态环境中微生物群落的形成和生态功能提供了基础信息。     

Characterization of microbial contamination in United States Air Force aviation fuel tanks

Bacteria and fungi, isolated from United States Air Force (USAF) aviation fuel samples, were identified by gas chromatograph fatty acid methyl ester (GC-FAME) profiling and 16S or 18S rRNA gene sequencing. Thirty-six samples from 11 geographically separated USAF bases were collected. At each base, an above-ground storage tank, a refueling truck, and an aircraft wing tank were sampled at the lowest sample point, or sump, to investigate microbial diversity and dispersion within the fuel distribution chain. Twelve genera, including four Bacillus species and two Staphylococcus species, were isolated and identified. Bacillus licheniformis, the most prevalent organism isolated, was found at seven of the 11 bases. Of the organisms identified, Bacillus sp., Micrococcus luteus, Sphinogmonas sp., Staphylococcus sp., and the fungus Aureobasidium pullulans have previously been isolated from aviation fuel samples. The bacteria Pantoea ananatis, Arthrobacter sp., Alcaligenes sp., Kocuria rhizophilia, Leucobacter komagatae, Dietza sp., and the fungus Discophaerina fagi have not been previously reported in USAF aviation fuel. Only at two bases were the same organisms isolated from all three sample points in the fuel supply distribution chain. Isolation of previously undocumented organisms suggests either, changes in aviation fuel microbial community in response to changes in aviation fuel composition, additives and biocide use, or simply, improvements in isolation and identification techniques.     

Chilling and cultivar type affect the diversity of bacterial endophytes colonizing sweet pepper (Capsicum anuum L.)

A climate chamber experiment was conducted to assay the effect of low temperatures (chilling) on the diversity of bacteria colonizing the endospheres of two thermophilic sweet pepper (Capsicum anuum L.) cultivars, Milder Spiral and Ziegenhorn Bello. Structural diversity was analyzed by 16S rRNA-based terminal restriction fragment length polymorphism (T-RFLP) analysis and by the generation of 16S rRNA gene libraries to determine dominant community members in T-RFLP profiles. Cultivable community members colonizing lines Milder Spiral and Ziegenhorn Bello were identified by 16S rRNA gene analysis. T-RFLP profiles and 16S rRNA gene libraries revealed a high heterogeneity of community composition due to chilling and suggested further the existence of cultivar-specific communities. The majority of isolates obtained from the cultivar Milder Spiral were assigned as high-G+C Gram-positive bacteria (Microbacterium sp., Micrococcus sp., Rhodococcus sp.) and Firmicutes (Staphylococcus sp.). Of the isolated endophytes obtained from cultivar Zeigenhorn Bello, 93% were affiliated with Staphylococcus aureus and Bacillus sp. (Firmicutes). The experimental set-up was suited to demonstrate that chilling and cultivar type can influence the diversity of bacterial endophytes colonizing sweet pepper. We propose additional chilling experiments to investigate the effect of chilling on functional, plant-beneficial abilities of bacterial endophytes associated with low-temperature-sensitive crops, such as sweet pepper.     

The effects of Cercospora leaf disease on the growth of groundnuts (Arachis hypogaea) in Nigeria

Three experiments with five alternately-branched and one sequentially-branched cultivar of groundnuts are described. Spraying to control Cercospora leaf spot disease doubled the leaf area duration and increased the total weight of a plant by about 65 %. Spraying increased the weight of kernels per plant but in the alternately-branched cultivars also increased the weight of stem and leaves. The proportion of the total dry weight in the kernels in the alternate cultivars was hardly changed by spraying. The sequentially-branched cultivar gave the same increase in total dry weight when sprayed but spraying resulted in a larger increase in the dry weight of kernels. The sequentially-branched cultivar, with few initials for vegetative growth, did not give the same increase in leaf growth.     

Detection of a Bacterium Associated with a Leaf Spot Disease of Maize in Brazil

A leaf spot disease of maize occurring in Brazil in the 1980s was described as being caused by the ascomycete Phaeosphaeria maydis (P. Henn) Rane. Payak and Renfro (imperfect form Phyllosticta sp.). Disease symptoms were dark-green water-soaked spots that later became necrotic lesions. There are no reports at present in the literature of re-infection by the fungus under controlled conditions, casting doubt on the true identity of the pathogen. In this study, cytological analyses of lesions at the initial stages did not detect the presence of fungal structures. Bacterial colonies with yellow pigmentation were isolated from the lesions, which reacted positively in hypersensitivity tests in tobacco plants. Maize plants were inoculated with the isolated bacteria. After 72 h incubation in a dew chamber, plants were transferred to a greenhouse, where they remained until evaluation. Typical symptoms of the disease were observed 5–7 days after inoculation of plants, only on treatments inoculated with the bacteria. The bacterium was re-isolated, which suggests its involvement in the initial phases of disease. The bacterium was identified as Pantoea ananas (synonym Erwinia ananas ).     

Detection and Molecular Diversity of Pantoea ananatis Associated with White Spot Disease in Maize,Sorghum and Crabgrass in Brazil

Bacteria of the genus Pantoea have become important plant pathogens worldwide in recent years. Pantoea ananatis was reported as the cause of maize white spot, a serious maize disease in Brazil, causing significant yield losses. However, very little information is available about how to detect this pathogen, its genetic variability and the putative alternative hosts in maize‐growing areas. To address these issues, we implemented a rapid and efficient PCR‐based method to identify P. ananatis isolated from leaves showing white spot symptoms and evaluated its genetic diversity in maize, sorghum and crabgrass. Of the 29 bacteria isolated from typical water‐soaked lesions of white spot disease that produced yellow colonies, 15 isolates were identified as P. ananatis by 16S rDNA sequencing and correctly detected by the PCR reaction, amplifying a specific fragment of the ice nucleation gene (ina). These P. ananatis isolates included 13 from maize, one from sorghum and one from crabgrass, while the other 14 yellow colony isolates were from other bacterial species, including two Pantoea species (Pantoea dispersa and Pantoea agglomerans) that were not amplified by the ina primers. These results indicate that the optimized PCR assay can be used to detect P. ananatis isolated from white spot lesions and could be used as a large‐scale and cost‐effective method of detecting this pathogen in leaf lesions on maize and other grasses. All isolates were evaluated for hypersensitive response (HR) on tobacco, revealing that some P. ananatis were able to induce HR. The high genetic variability revealed by rep‐PCR did not differentiated the P. ananatis isolates based on their hosts or HR reaction. The detection, characterization and diversity of P. ananatis from maize, sorghum and crabgrass in our study can be applied in understanding epidemiology and designing control strategies for maize white spot disease in Brazil.     

Effects of growth-promoting rhizobacteria on maize growth and rhizosphere microbial community under conservation tillage in Northeast China

Conservation tillage in conjunction with straw mulching is a sustainable agricultural approach. However, straw mulching reduces the soil temperature, inhibits early maize growth and reduces grain yield in cold regions. To address this problem, we investigated the effects of inoculation of plant growth-promoting rhizobacteria (PGPR) on maize growth and rhizosphere microbial communities under conservation tillage in Northeast China. The PGPR strains Sinorhizobium sp. A15, Bacillus sp. A28, Sphingomonas sp. A55 and Enterobacter sp. P24 were isolated from the maize rhizosphere in the same area and inoculated separately. Inoculation of these strains significantly enhanced maize growth, and the strains A15, A28 and A55 significantly increased grain yield by as much as 22%–29%. Real-time quantitative PCR and high-throughput sequencing showed that separate inoculation with the four strains increased the abundance and species richness of bacteria in the maize rhizosphere. Notably, the relative abundance of Acidobacteria_Subgroup_6, Chloroflexi_KD4-96, and Verrucomicrobiae at the class level and Mucilaginibacter at the genus level were positively correlated with maize biomass and yield. Inoculation with PGPR shows potential for improvement of maize production under conservation tillage in cold regions by regulating the rhizosphere bacterial community structure and by direct stimulation of plant growth.     

板栗干腐病研究:Ⅱ.症状及病原

对板栗干腐病症状及病原的研究结果表明:1.板栗干腐病主要发生于种仁上,其病斑大小、质地不一。按色泽可分为褐、黑、白、灰、黄五类。果实其它部位也偶有症状产生;2.此病为多种真菌复合侵染所致。主要的致病菌有Phoma、Phomopsis、Dothiorella、Gloeosporium和Cytospora等属真菌,另有十余种弱寄生及腐生菌有时也混合发生。     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Phenotypic and genetic diversity of rice seed-associated bacteria and their role in pathogenicity and biological control

Aims:  To study the phenotypic and genetic diversity of culturable bacteria associated with rice seed and to asses the antagonistic and pathogenic potential of the isolated bacteria.
Methods and Results:  Seed of rice cultivar PSBRc14 was collected from farmers' fields of irrigated lowland in southern Luzon, Philippines. Isolations of distinct colonies yielded 498 isolates. Classification of the isolates according to similarities in cellular characteristics, whole-cell fatty acid composition, and colony appearance differentiated 101 morphotype groups. Predominant bacteria were Coryneform spp., Pantoea spp. and Pseudomonas spp. Other bacteria regularly present were Actinomycetes spp., Bacillus pumilus , B. subtilis , Burkholderia glumae , Enterobacter cloacae , Paenibacillus polymyxa , Staphylococcus spp. and Xanthomonas spp. The genetic diversity among isolates was assessed by BOX-PCR fingerprinting of genomic DNA and represented 284 fingerprint types (FPTs). Most FPTs (78%) were not shared among samples, while eight FPTs occurred frequently in the samples. Seven of these FPTs also occurred frequently in a previous collection made from rainfed lowlands of Iloilo island, Philippines. Sixteen per cent of the isolates inhibited in vitro the mycelial growth of the rice pathogens Rhizoctonia solani and Pyricularia grisea , whereas 4% were pathogens identified as Burkholderia glumae , Burkholderia gladioli and Acidovorax avenae ssp. avenae .
Conclusions:  This study reveals a broad morphological and genetic diversity of bacteria present on seed of a single rice cultivar.
Significance and Impact of the Study:  This line of work contributes to a better understanding of the microbial diversity present on rice seed and stresses its importance as a carrier of antagonists and pathogens.     

柑橘黄龙病赣南脐橙内生菌种群结构分析

【目的】分析赣南脐橙黄龙病植株和健康植株叶片内生菌,对比不同培养条件下培养出的内生菌,为筛选出对柑橘黄龙病原菌有影响的伴生菌奠定理论基础。【方法】通过PCR方法对脐橙中黄龙病菌进行验证,并基于16S r RNA基因高通量测序技术对患病与健康赣南脐橙叶片内生菌以及不同培养基富集培养后的内生菌进行多样性分析。【结果】所采集样品中有5株患病株,5株健康株。5株病株中共同含有的细菌属有13个,其中7个在5株健株中也共同存在。Defluviicoccus属和Granulicella属在病健株植物中都是优势菌属,且在健株中的平均含量高于病株。病株与健株的样品相似度存在明显界线。富集培养后不同样本和不同培养基中菌属分布不同。肠杆菌属(Enterobacter)、短小杆菌属(Curtobacterium)、假单胞菌属(Pseudomonas)和泛菌属(Pantoea)得到了大量富集,不动杆菌属和沙雷氏菌属等9个菌属富集量较少。另外,培养和未培养各样本间未分类菌(Unclassified)含量差异也较大。【结论】赣南脐橙患病植株和健康植株叶片内生菌有着明显差异,黄龙病菌的存在改变了脐橙叶片原有内生细菌的菌群结构。从活体植物组织内直接检测才能得到真正的植物内生菌群落分布情况。通过分析菌群的差异,有望找到与柑橘黄龙病菌生长相关的伴生菌。