Pine wilt disease and possible causes of its incidence in Russia

Surveys of forests and stockpiled timber of pine, spruce, larch, and silver fir in 14 administrative subjects of the Russian Federation revealed widespread occurrence of the coniferous wood parasitic nematode Bursaphelenchus mucronatus. Twenty species of bacteria belonging to 13 genera have been detected in 25 B. mucronatus isolates, and their identity has been determined by direct sequencing of the 16S RNA gene. The most frequently occurring were bacteria from the genera Pseudomonas, Stenotrophomonas, Pantoea, Bacillus, Burkholderia, and Serratia. Prevalence of Pseudomonas brenneri and P. fluorescence, which were also found in the nematode dauer larva (L_IV) isolated from the fir sawyer beetle Monochamus urussovi, have also been assessed. Two nematode B. xylophilus isolates from Portugal and one isolate from the United States have been examined, and 10 symbiotic bacteria species have been isolated, including Agrobacterium tumefacience, P. fluorescens, P. brenneri, Rahnella aquatilis, Stenotrophomonas maltophilia, S. rhizophila, and Yersinia mollaretii.     

Joint Influence of Pratylenchus penetrans (Nematoda) and Leptinotarsa decemlineata (Insecta) on Solanum tuberosum Productivity and Pest Population Dynamics

The joint action of a plant parasitic nematode, Pratylenchus penetrans (root-lesion nematode), and an insect defoliator, Leptinotarsa decemlineata (Colorado potato beetle), on growth, development, and yield of Solanum tuberosum cv. Superior was studied in the field. Three population densities of P. penetrans were superimposed on each of three population levels of L. decemlineata. The major impact of P. penetrans on final yield was through a reduction in the number of tubers formed during tuber initiation. Defoliation by L. decemlineata increased with time as larvae advanced through successive instars and densities increased. This resulted in a significant reduction in tuber weight and numbers. Total yield of S. tuberosum was decreased by 66% with increasing population densities of L. decemlineata and 27 % with increasing densities of P. penetrans. L. decemlineata feeding did not affect soil population densities of P. penetrans. Root population densities of P. penetrans, however, were significantly (P = 0.05) higher in plants maintained beetle free than in plants grown in the presence of the beetles.     

Diversity of culturable root-associated/endophytic bacteria and their chitinolytic and aflatoxin inhibition activity of peanut plant in China

A total of 72 isolates of root-associated/endophytic (RAE) bacteria were isolated from peanut plants grown in the main producing areas of 6 provinces in China. The 16S rRNA gene sequences of these isolates were determined and phylogenetic analyses revealed that 72 isolates belonged to the classes Bacilli (49 isolates) and Gammaproteobacteria (23 isolates). The majority of RAE bacteria in Bacilli belonged to 2 genera, Bacillus and Lysinibacillus (48 and 1) while those in Gammaproteobacteria belonged to the genera Enterobacter, Serratia, Stenotrophomonas, and Pseudomonas (7, 11, 3 and 2 isolates, respectively). This is the first report of Lysinibacillus xylanilyticus isolate as biocontrol agent against AFs. All of the selected RAE bacteria showed inhibitory activities against Aspergillus parasiticus (A. parasiticus) growth and/or aflatoxins (AFs) production by visual agar plate assay and tip culture method. Most of the RAE bacteria strains (96?% strains) were determined to have decreased mycelia growth or AFs production levels by >50?% (p?<?0.05). Bacterial isolates were further characterized for chitinolytic activity and 22 strains (30?% strains) of identified RAE bacteria degraded colloidal chitin on the chitin medium plate. Ten selected chitinolytic RAE bacteria were tested for antifungal activity on peanuts and most of them significantly decreased mycelial growth and AFs production levels by >90?%. These results showed a wide distribution of biological control bacteria against AFs in Chinese peanut main producing areas and the selected RAE bacteria could potentially be utilized for the biocontrol of toxicogenic fungi.     

Diversity of sporadic symbionts and nonsymbiotic endophytic bacteria isolated from nodules of woody, shrub, and food legumes in Ethiopia

Fifty-five bacterial isolates were obtained from surface-sterilized nodules of woody and shrub legumes growing in Ethiopia: Crotalaria spp., Indigofera spp., and Erythrina brucei, and the food legumes soybean and common bean. Based on partial 16S rRNA gene sequence analysis, the majority of the isolates were identified as Gram-negative bacteria belonging to the genera Achromobacter, Agrobacterium, Burkholderia, Cronobacter, Enterobacter, Mesorhizobium, Novosphingobium, Pantoea, Pseudomonas, Rahnella, Rhizobium, Serratia, and Variovorax. Seven isolates were Gram-positive bacteria belonging to the genera Bacillus, Paenibacillus, Planomicrobium, and Rhodococcus. Amplified fragment length polymorphism (AFLP) fingerprinting showed that each strain was genetically distinct. According to phylogenetic analysis of recA, glnII, rpoB, and 16S rRNA gene sequences, Rhizobium, Mesorhizobium, and Agrobacterium were further classified into six different genospecies: Agrobacterium spp., Agrobacterium radiobacter, Rhizobium sp., Rhizobium phaseoli, Mesorhizobium sp., and putative new Rhizobium species. The strains from R. phaseoli, Rhizobium sp. IAR30, and Mesorhizobium sp. ERR6 induced nodules on their host plants. The other strains did not form nodules on their original host. Nine endophytic bacterial strains representing seven genera, Agrobacterium, Burkholderia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, and Serratia, were found to colonize nodules of Crotalaria incana and common bean on co-inoculation with symbiotic rhizobia. Four endophytic Rhizobium and two Agrobacterium strains had identical nifH gene sequences with symbiotic Rhizobium strains, suggesting horizontal gene transfer. Most symbiotic and nonsymbiotic endophytic bacteria showed plant growth-promoting properties in vitro, which indicate their potential role in the promotion of plant growth when colonizing plant roots and the rhizosphere.     

Heterologous Expression of Bacillus thuringiensis Vegetative Insecticidal Protein-Encoding Gene vip3LB in Photorhabdus temperata Strain K122 and Oral Toxicity against the Lepidoptera Ephestia kuehniella and Spodoptera littoralis

Photorhabdus temperata and Bacillus thuringiensis are entomopathogenic bacteria exhibiting toxicities against different insect larvae. Vegetative Insecticidal Protein Vip3LB is a Bacillus thuringiensis insecticidal protein secreted during the vegetative growth stage exhibiting lepidopteran specificity. In this study, we focused for the first time on the heterologous expression of vip3LB gene in Photorhabdus temperata strain K122. Firstly, Western blot analyses of whole cultures of recombinant Photorhabdus temperata showed that Vip3LB was produced and appeared lightly proteolysed. Cellular fractionation and proteinase K proteolysis showed that in vitro-cultured recombinant Photorhabdus temperata K122 accumulated Vip3LB in the cell and appeared not to secrete this protein. Oral toxicity of whole cultures of recombinant Photorhabdus temperata K122 strains was assayed on second-instar larvae of Ephestia kuehniella, a laboratory model insect, and the cutworm Spodoptera littoralis, one of the major pests of many important crop plants. Unlike the wild strain K122, which has no effect on the larval growth, the recombinant bacteria expressing vip3LB gene reduced or stopped the larval growth. These results demonstrate that the heterologous expression of Bacillus thuringiensis vegetative insecticidal protein-encoding gene vip3LB in Photorhabdus temperata could be considered as an excellent tool for improving Photorhabdus insecticidal activities.     

Differential pathogenicity of two entomopathogenic bacteria,Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle,Tribolium castaneum

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae, respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A₂ activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.     

Rhizobacterial Volatiles Affect the Growth of Fungi and Arabidopsis thaliana

Volatiles of Stenotrophomonas, Serratia, and Bacillus species inhibited mycelial growth of many fungi and Arabidopsis thaliana (40 to 98%), and volatiles of Pseudomonas species and Burkholderia cepacia retarded the growth to lesser extents. Aspergillus niger and Fusarium species were resistant, and B. cepacia and Staphylococcus epidermidis promoted the growth of Rhizoctonia solani and A. thaliana. Bacterial volatiles provide a new source of compounds with antibiotic and growth-promoting features.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

Meloidogyne incognita Infested Soil Amended With Chicken Litter

The effects of chicken litter on Meloidogyne incognita in cotton, Gossypium hirsutum cv. DPL50 were determined in field microplots. Litters (manure and pine-shaving bedding) from a research facility and a commercial broiler house were used. Treatments consisted of 0.25%, 0.5%, and 1% litter by dry weight of soil for each kind of litter. Three control treatments consisted of soil not amended with litter, with and without nematodes, and one treatment to which mineral fertilizer was added at a nitrogen rate equivalent to that of the 0.5% litter rate, with nematodes. Microplots were inoculated at planting with 900 eggs/100 cm³ soil in 1993 and 1,000 eggs/100 cm³ soil in 1994. At 92 and 184 days after planting, nematode population densities decreased linearly with increasing rates of litter. Nematode numbers at midseason were larger in plots treated with mineral fertilizer than in plots treated with a rate of litter equivalent to the 0.5% rate. Fungal and bacterial population densities fluctuated throughout the growing season. Bacterial numbers had a positive linear relationship, with increasing rates of litter only in October 1993; however, significant positive relationships were observed throughout the 1994 growing season. In 1994, nematode population density at 92 days after planting decreased linearly with increasing bacterial numbers 30 days after planting. No other significant relationships between nematode densities and microbial densities were observed. Fungi and bacteria isolated from the litter and litter-amended soil were identified. Fungal genera isolated included Acremonium, Aspergillus, Eurotium, Paecilomyces, Petriella, and Scopulariopsis, whereas bacteria genera included Arthrobacter, Bacillus, and Pseudomonus.     

Genetic diversity of phosphate-solubilizing peanut (Arachis hypogaea L.) associated bacteria and mechanisms involved in this ability

In this study, attempts were made to analyze mechanisms involved in the bacterial phosphate-solubilizing ability of peanut isolates. Bacteria were taxonomically identified by analysis of 16S rDNA sequence. Levels of soluble P released by the isolates in unbuffered or buffered with Tris–HCl or MES NBRIP-BPB medium as well as the production of D-gluconic acid were determined in their culture. Presence of two of the genes encoding the cofactor PQQ of GDH enzyme was analyzed in the genome of this bacterial collection. 16S rDNA sequence analysis indicated that isolates belong to genera Serratia, Enterobacter, Pantoea, Acinetobacter, Bacillus and Enterococcus. All bacteria showed ability to solubilize tricalcium phosphate either in unbuffered or buffered medium. Nevertheless, addition of buffer solutions reduced levels of Pi liberated by the isolates. Although almost all isolates produced detectable amounts of D-gluconic acid, no correlation with levels of P soluble released were observed. The presence of pqqE and pqqC genes was detected only in Gram negative bacteria. It was concluded from this study that the mechanism involved in phosphate solubilization is organic acids production and, presence of pqq genes in all Gram negative bacteria analyzed encourages to confirm their role in bacterial phosphate solubilizing ability as well to identify genes involved in this PGP trait in Gram positive bacteria.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

Laboratory Evaluation of Isaria fumosorosea CCM 8367 and Steinernema feltiae Ustinov against Immature Stages of the Colorado Potato Beetle

The Colorado potato beetle, Leptinotarsa decemlineata, has developed resistance to most registered pesticides and has become one of the most difficult insect pests to control. Development of new biopesticides targeting this pest might solve the resistance problem and contribute to sustainable crop production. Laboratory experiments were conducted to assess the efficacy of Isaria fumosorosea (syn. Paecilomyces fumosoroseus) strain CCM 8367 against L. decemlineata when applied alone or combined with the entomopathogenic nematode Steinernema feltiae. The last-instar larvae of the Colorado potato beetle showed the highest susceptibility to I. fumosorosea followed by pre-pupae and pupae. The median lethal concentration (LC₅₀) was estimated to be 1.03×10⁶ blastospores/ml. The strain CCM 8367 was more virulent, causing 92.6% mortality of larvae (LT₅₀ = 5.0 days) compared to the reference strain Apopka 97, which caused 54.5% mortality (LT₅₀ = 7.0 days). The combined application of the fungus with the nematodes increased the mortality up to 98.0%. The best results were obtained when S. feltiae was applied simultaneously with I. fumosorosea (LT₅₀ = 2.0 days); later application negatively affected both the penetration rate and the development of the nematodes. We can conclude that the strain CCM 8367 of I. fumosorosea is a prospective biocontrol agent against immature stages of L. decemlineata. For higher efficacy, application together with an entomopathogenic nematode is recommended.     

Survey of Plant Drought-Resistance Promoting Bacteria from Populus euphratica Tree Living in Arid Area

Two hundred and thirty-two bacterial strains were isolated from the rhizospheric soil of Populus euphratica which is the dominant tree living in extreme arid regions in northwest China. Some strains with plant growth-promoting bacteria related metabolic characteristics were able to promote drought resistance in plants after inoculation. Ten strains with the greatest effects increased the dry weight of wheat shoots from 0.5 to 34.4 %, and the surface area of the root systems from 12.56 to 212.17 % compared to the control after drought treatment whereas no obvious promoting effect was observed in normal water conditions. These 10 strains were identified to be of the genera Pseudomonas, Bacillus, Stenotrophomonas and Serratia by 16S rRNA (rrs) gene sequence alignment. Among these strains, Serratia sp. 1-9 and Pseudomonas sp. 5-23 were the two most effective strains. Both of them produced auxin and the production increased significantly when cultured under simulated drought conditions which are inferred to be the most plausible mechanism for their plant growth-promoting effect under drought stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0479-3) contains supplementary material, which is available to authorized users.     

Lethal and sublethal effects of Iranian isolates of Steinernema feltiae and Heterorhabditis bacteriophora on the Colorado potato beetle, Leptinotarsa decemlineata

To determine the LC₅₀ values of two entomopathogenic nematodes against Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae) prepupae, different concentrations of the nematodes were tested in soil. Because of the different temperature requirements of the two nematode species, bioassay experiments were conducted at 20 ± 1°C and 27 ± 2°C for Steinernema feltiae Filipjev (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae), respectively. Both the isolates were effective against L. decemlineata. LC₅₀ values of H. bacteriophora against progeny of field-collected adults and laboratory-reared adults were estimated as 8.5 and 7.6 IJ per prepupa, respectively. For S. feltiae the value was calculated as 51.2 IJ per prepupa against offspring of laboratory-reared adults of L. decemlineata only. Cellular encapsulation of both nematode species was observed. Sublethal nematode concentrations caused wing deformation and delayed metamorphosis which may affect Colorado potato beetle adult fitness.     

Bacterial Community Diversity in the Brazilian Atlantic Forest Soils

The aim of this study was to characterize the bacterial community diversity of the Brazilian Atlantic forest soil by means of both cultivation and 16S rRNA clone libraries. A collection of 86 representative isolates, obtained from six samples of Atlantic forest soils from the National Park of Serra dos Órgãos (PARNASO), belonged to the genera Arthrobacter, Bacillus, Burkholderia, Leifsonia, Paenibacillus, Pseudomonas, Ralstonia, Serratia, and Streptomyces according to the 16S rRNA sequences. Representative isolates from the different genera degraded cellulose and lignin. The culture-independent analysis based on 894 partial 16S rRNA gene sequences revealed that the most frequently retrieved groups belonged to the phyla Acidobacteria (29–54%), Proteobacteria (16–38%), and Verrucomicrobia (0.6–14%). The majority of the sequences (82.6%) were unidentified singletons and doubletons, indicating a high diversity of rare unique sequences. Chao1 estimator disclosed a high number of phyla (41–152) and species (263–446). This is the first survey on the Atlantic Forest soils using a combination of cultivation and culture-independent approaches. We conclude that the Brazilian Atlantic Forest soil represents a vast source of novel bacteria.     

Antibacterial compounds in estuarine submersed aquatic plants

Estuarine seagrasses often exist in nutrient-rich waters and thus might benefit from mechanisms to control exterior growth of epiphytic microorganisms. In this study, we tested extracts from three estuarine seagrass species, Potamogeton pectinatus L. (sago pondweed), Potamogeton perfoliatus L. (redhead grass) and Ruppia maritima L. (wigeon grass), for antibacterial activity. Methanolic extracts of axenic cultured plants were effective against all gram-positive bacteria tested, inhibiting growth of 12 species in the genera Micrococcus, Staphylococcus, Streptococcus, Bacillus, Aerococcus, Mycobacterium and Corynebacterium. Some gram-negative species in the genera Vibrio, Listonella and Pasteurella were also sensitive. Other gram-negative bacteria were resistant. Antibacterial activity was detected in field-collected plants in spring, but appeared absent in plants collected in fall. These seagrass species thus appear to possess an antibacterial agent. It has not been identified and may consist of one or multiple compounds. The antibacterial agent in P. pectinatus was released into the water column in vitro at concentrations sufficient to inhibit or kill sensitive bacterial species. The agent was stable, with an in vitro half-life of 12 days at 25 °C and 6.6 days at 37 °C. It is possible that antibacterial production has ecological effects upon the bacterial and faunal communities associated with seagrass ecosystems.     

Differential sensitivity of plant-associated bacteria to sulfonylurea and imidazolinone herbicides

The side effects of sulfonylurea and imidazolinone herbicides on plant-associated bacteria were investigated under pure culture conditions. Eighteen isolates, belonging to the genera Azotobacter, Azospirillum, Bacillus, Enterobacter Pseudomonas and Serratia, were exposed to four active compounds at concentration ranges similar to those in field soil. The sulfonylureas chlorsulfuron and rimsulfuron inhibited the growth of one of two Azospirillum and one of four Pseudomonas strains, while the imidazolinones imazapyr and imazethapyr were effective on two out of five Bacillus isolates. Surfactants in commercial formulation significantly enhanced rimsulfuron toxicity. With the exception of one Azospirillum strain, the differential tolerance of rhizobacteria to these herbicides was related to a differential sensitivity of their target, the activity of the first enzyme in branched-chain amino acid biosynthesis, acetohydroxyacid synthase (AHAS).Greenhouse pot studies were performed to assess the occurrence of inhibitory effects on bacterial growth in field conditions. Maize seedlings were bacterized with the two strains which had shown in vitro sensitivity to sulfonylureas. Following the application to the soil of a commercial formulation of rimsulfuron at rates of 0, 0.2 and 0.5 mol a.i. kg^–1, significative differences in the resulting degree of bacterial root colonization were found. Moreover, upon co-inoculation with two strains, one tolerant and one sensitive to the herbicide, the presence of rimsulfuron significantly enhanced root occupancy by resistant bacteria, suggesting that shifts in the microbial community structure of crop rhizosphere could indeed result as a consequence of weed control by AHAS inhibitors.Abbreviations AHAS acetohydroxyacid synthase - CETAB cetyltrimethylammonium bromide - ID₅₀ concentration causing 50% inhibition of enzyme activity - LD₅₀ concentration causing 50% decrease of growth constant value     

Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.