Occurrence,physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants

Endophytic diazotrophic bacteria could be isolated from the energy plants Pennisetum purpureum, Miscanthus sinensis, Miscanthus sacchariflorus and Spartina pectinata using semisolid nitrogen free media. Higher levels of diazotrophic bacteria were found if no nitrogen fertilizer was applied. The bacteria were characterized on the basis of typical morphology, physiological tests, and the use of phylogenetic oligonucleotide probes. They belong partially to the species Azospirillum lipoferum and Herbaspirillum seropedicae while others supposedly represent a new species of Herbaspirillum. Using PCR-fingerprinting techniques a limited genetic diversity of these isolates was found which may indicate an adaptation to the specific conditions of the interior of these plants.     

16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Microbial control of arthropod pests of tropical tree fruits

A multitude of insects and mites attack fruit crops throughout the tropics. The traditional method for controlling most of these pests is the application of chemical pesticides. Growing concern on the negative environmental effects has encouraged the development of alternatives. Inundatively and inoculatively applied microbial control agents (virus, bacteria, fungi, and entomopathogenic nematodes) have been developed as alternative control methods of a wide variety of arthropods including tropical fruit pests. The majority of the research and applications in tropical fruit agroecosystems has been conducted in citrus, banana, coconut, and mango. Successful microbial control initiatives of citrus pests and mites have been reported. Microbial control of arthropod pests of banana includes banana weevil, Cosmopolites sordidus Germar (Coleoptera: Curculionidae) (with EPNs and fungi) among others Oryctes rhinoceros (L.) is one of the most important pests of coconut and one of the most successful uses of non-occluded virus for classical biological control. Key pests of mango that have been controlled with microbial control agents include fruit flies (Diptera: Tephritidae) (with EPNs and fungi), and other pests. Also successful is the microbial control of arthropod pests of guava, papaya and pineapple. The challenge towards a broader application of entomopathogens is the development of successful combinations of entomopathogens, predators, and parasitoids along with other interventions to produce effective and sustainable pest management.     

Genetic transformation of perennial tropical fruits

Summary Genetic transformation provides the means for modifying single horticultural traits in perennial plant cultivars without altering their phenotype. This capability is particularly valuable for perennial plants and tree species in which development of new cultivars is often hampered by their long generation time, high levels of heterozygosity, nucellar embryony, etc. Most of these conditions apply to many tropical and subtropical fruit crops. Targeting specific gene traits is predicated upon the ability to regenerate elite selections of what are generally trees from cell and tissue cultures. The integrity of the clone would thereby remain unchanged except for the altered trait. This review provides an overview of the genetic transformation of perennial tropical and subtropical fruit crops, i.e., citrus (Citrus spp.), banana and plantain (Musa groups AAA, AAB, ABB, etc.), mango (Mangifera indica L.), pineapple (Ananas comosus L.), avocado (Persea americana Mill.), passion fruit (Passiflora edulis L.), longan (Dimocarpus longan Lour.), and litchi (Litchi chinensis Sonn.).     

Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.     

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.     

16S Ribosomal DNA Characterization of Nitrogen-Fixing Bacteria Isolated from Banana (Musa spp.) and Pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10² to 4.6×10²/ml for seawater, 4.0×10⁴ to 4.3×10⁵/g wet wt for eelgrass-bed sediment, and 2.1 × 10⁵ to 1.2 × 10⁷/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.     

Natural occurrence of Azospirillum brasilense in strawberry plants

Azospirillum species are free-living nitrogen-fixing bacteria commonly found in soil and in association with roots of different plant species. For their capacity to stimulate growth they are known as plant growth-promoting bacteria (PGPB). In this work, we demonstrate the natural occurrence and colonization of different parts of strawberry plants by Azospirillum brasilense in the cropping area of Tucumán, Argentina. Although bacteria isolations were carried out from two strawberry cultivars, e.g., Camarosa and Pájaro, attempts were successful only with the cultivar Camarosa. Whereas different strains of Azospirillum were isolated from the root surface and inner tissues of roots and stolons of the cultivar Camarosa, we have not obtained Azospirillum isolates from the cultivar Pájaro. After microbiological and molecular characterization (ARDRA) we determined that the isolates belonged to the species A. brasilense. All isolates showed to have the capacity to fix nitrogen, to produce siderophores and indoles. Local isolates exhibited different yields of indoles production when growing in N-free NFb semisolid media supplemented or not with tryptophan (0.1 mg ml⁻¹). This is the first report on the natural occurrence of A. brasilense in strawberry plants, especially colonizing inner tissues of stolons, as well as roots. The local isolates showed three important characteristics within the PGPB group: N₂-fixation, siderophores, and indoles production.     

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

Pineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.     

Azospirilli of Uzbekistan Soils and their Influence on Growth and Development of Wheat Plants

Associative nitrogen-fixing bacteria have been isolated, which were related to Azospirillum genus, by their morphological–cultural and physiological–biochemical ability to grow in microaerophilic conditions, as well as by a number of phenotypic traits. They comprised two species, namely, Azospirillum brasilense and Azospirillum lipoferum. Azospirilli strains displayed a varying salt resistance on potato medium containing a range of NaCl concentrations from 100 to 800 mM. The decrease in the nitrogen-fixing activity of azospirilli was detected starting from 200 mM NaCl. The biomass of the inoculated local varieties of wheat, Unumdor Bugdoi and Karlik 85, in microvegetation experiments exceeded the biomass of control plants by 20–50%. During the vegetation, some azospirilli strains formed spontaneous nodules on the wheat roots.     

Tatumella ptyseos,an Unrevealed Causative Agent of Pink Disease in Pineapple

Pink disease is a major problem in the pineapple canning industry. Affected fruit acquire a brownish pigment after pasteurization and can contaminate non‐affected fruit before they are released to the consumer. In the last few years, Pantoea citrea has been described as the causative agent of pink disease. In this study, over 300 bacterial isolates from pineapple plants, growing in Mexican commercial fields, and from soil close to plant roots were recovered. Over 250 isolates showed a very high similarity in their phenotypic and genotypic traits with Tatumella ptyseos, a close relative of Pantoea. These isolates exhibited typical pathogenicity reactions in pineapple juice tests, pineapple slices and fruit. On this basis, molecular identification procedures for the Tatumella isolates associated with pink disease were implemented. In affected fruit populations around 10⁶ CFU/g of fresh tissue were recovered. This is first time that T. ptyseos is demonstrated as a causal agent of pink disease.     

THE COLIFORM BACTERIA ASSOCIATED WITH POTATO BLACK-LEG AND OTHER SOFT ROTS

Twelve glasshouse trials using twenty-five isolates of coliform soft-rot bacteria obtained from various countries showed that all the isolates produced typical black-leg of potato at temperatures of 76° F. (24.5° C.) or over, while some of them could also produce the disease at temperatures below 66° F. (19° C.).
Thus the isolates fall into two main groups of which the high-temperature group originates mostly from tropical or subtropical countries or from plants grown in heated glasshouses, whereas the low-temperature group is indigenous in the north temperate regions. The high-temperature group comprises Pectobacterium carotovorum (Erwinia carotovora), P. carotovorum var. chrysanthemi (E. chrysanthemi) and P. carotovorum var. aroideae (E. aroideae). The low-temperature group consists of one variety, P. carotovorum var. atrosepticum (E. atroseptica).
This temperature relation provides a possible means of discovering the origin of the black-leg pathogen in crops grown from Scottish seed in certain tropical or subtropical countries.
As a result of this investigation it is considered that all soft-rot coliform bacteria are varieties of a single species, P. carotovorum (Jones) Waldee.     

Towards better use of Indonesian peatlands with paludiculture and low-drainage food crops

The current drainage-based peatland management systems in Indonesia result in high fire risks, soil subsidence and CO₂ emissions. This study aims to assess different alternatives of peatland crops in order to help prevent further degradation of peatlands in Indonesia. We focus on tropical peatland crops that provide food and that are of particular interest to smallholders. We compare various peatland food crops that are commonly grown with no drainage (paludiculture) or drainage below 50 cm in our study area, Central Kalimantan, Indonesia in terms of sustainability, profitability, scalability of the market and acceptability to farmers. Our results show that sago (Metroxylon sagu), banana (Musa paradisiaca) and pineapple (Ananas comosus) followed by water spinach/kangkong (Ipomoea aquatica), kelakai/edible fern (Stenochlaena palustris), illipe nut/tengkawang (Shorea spp.), dragon fruit (Hylocereus undatus), mangosteen (Garcinia mangostana) and sweet melon/melon (Cucumis melo) are the best options based on the aggregated scores for these criteria (but precaution should be taken when planting crops that require low drainage). Sago palm and illipe nut have the highest scores for both sustainability and scalability of market, whereas banana, pineapple and sweet melon have the highest scores in term of the scalability of market and acceptability to farmers. We also address key opportunities and bottlenecks for the development of paludiculture food crops and present recommendations for the implementation of paludiculture in Indonesian peatlands.

     

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants

Abstract The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed. Received: 16 March 1999; Accepted: 27 August 1999; Online Publication: 23 February 2000     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Presence of endophytic diazotrophs in sugarcane juice

Summary In this work we investigated the diazotrophs present in the juice of different varieties of sugarcane. Samples of the aerial part of sugarcane, between 3 and 5 months old, were assessed for the presence of diazotrophs. The isolated nitrogen-fixing microorganisms were identified as Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and Azospirillum brasilense. These microorganisms use organic acids found in the sugarcane juice as an energy source. They also use sugars, such as free glucose, that are present in all the organs of the aerial part. The distribution of endophytic microorganisms in sugarcane depends on the different organic acid and sugar concentrations present during development and growth. We concluded that in more mature regions of the sugarcane stem Gluconacetobacter diazotrophicus grows more abundantly than Herbaspirillum seropedicae or Azospirillum brasilense.     

Production of indole-3-acetic acid and gibberellins A1 and A3 by Acetobacter diazotrophicus and Herbaspirillum seropedicae in chemically-defined culture media

The characterization by capillary gas chromatography-mass spectrometry of the plant hormones indole-3-acetic acid and the gibberellins GA₁ and GA₃ from chemically-defined cultures of Acetobacter diazotrophicus and Herbaspirillum seropedicae is reported. Both bacteria are endophytic in gramineae species where they promote growth and yield. Quantification was also done by selected ion monitoring with [17,17-²H₂]-Gibberellin A₁, [17,17-²H₂]-Gibberellin A₃ and [¹³C₆]-indole-3-acetic acid as internal standards. The results presented show the importance of studying phytohormonal production when the interrelationships between plants and microorganisms are analyzed and may help explain the beneficial effects of endophytic bacteria to the host plant, as has been demonstrated previously for Azospirillum spp.