Beneficial native bacteria improve survival and mycorrhization of desert truffle mycorrhizal plants in nursery conditions

Sixty-four native bacterial colonies were isolated from mycorrhizal roots of Helianthemum almeriense colonized by Terfezia claveryi, mycorrhizosphere soil, and peridium of T. claveryi to evaluate their effect on mycorrhizal plant production. Based on the phylogenetic analysis of the 16S rDNA partial sequence, 45 different strains from 17 genera were gathered. The largest genera were Pseudomonas (40.8 % of the isolated strains), Bacillus (12.2 % of isolated strains), and Varivorax (8.2 % of isolated strains). All the bacteria were characterized phenotypically and by their plant growth-promoting rhizobacteria (PGPR) traits (auxin and siderophore production, phosphate solubilization, and ACC deaminase activity). Only bacterial combinations with several PGPR traits or Pseudomonas sp. strain 5, which presents three different PGPR traits, had a positive effect on plant survival and growth. Particularly relevant were the bacterial treatments involving auxin release, which significantly increased the root-shoot ratio and mycorrhizal colonization. Moreover, Pseudomonas mandelii strain 29 was able to considerably increase mycorrhizal colonization but not plant growth, and could be considered as mycorrhiza-helper bacteria. Therefore, the mycorrhizal roots, mycorrhizosphere soil, and peridium of desert truffles are environments enriched in bacteria which may be used to increase the survival and mycorrhization in the desert truffle plant production system at a semi-industrial scale.     

<Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> HW2 enhanced cucumber resistance against cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of cucumber plants worldwide. In the present study, we use plant growth-promoting rhizobacteria (PGPR) to control this virus effectively. Stenotrophomonas maltophilia HW2 was isolated from healthy cucumber root, exhibited a good biocontrol efficacy against CGMMV. Here, it is documented that 20 d after virus inoculation, the biocontrol efficacy of HW2 reached 52.61%. HW2 can effectively colonize in cucumber rhizosphere, and also promoted cucumber plants growth. We also examined the effect of HW2 on viral replication and its mechanism. Compared with the control, HW2 pre-treated plants could delay virus replication for more than 3 d and inhibit viral protein genes (CP, MP, Rep) expression in the cucumber leaf. The expression of antioxidant enzyme genes (SOD and CAT) and defense-related genes (PR1 and PR5) were quickly induced by HW2. These results suggest that HW2 induced plant defense responses to CGMMV by increasing the expression of defense response genes. We report for the first time that Stenotrophomonas maltophilia improved cucumber resistance against CGMMV, which highlights the applying of PGPR on controlling of virus diseases.     

Endophytic microbes <Emphasis Type="Italic">Bacillus</Emphasis> sp. LZR216-regulated root development is dependent on polar auxin transport in <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Key message

Endophytic microbes Bacillus sp. LZR216 isolated from Arabidopsis root promoted Arabidopsis seedlings growth. It may be achieved by promoting the lateral root growth and inhibiting the primary root elongation.

Abstract

Plant roots are colonized by an immense number of microbes, including epiphytic and endophytic microbes. It was found that they have the ability to promote plant growth and protect roots from biotic and abiotic stresses. But little is known about the mechanism of the endophytic microbes-regulated root development. We isolated and identified a Bacillus sp., named as LZR216, of endophytic bacteria from Arabidopsis root. By employing a sterile experimental system, we found that LZR216 promoted the Arabidopsis seedlings growth, which may be achieved by promoting the lateral root growth and inhibiting the primary root elongation. By testing the cell type-specific developmental markers, we demonstrated that Bacillus sp. LZR216 increases the DR5::GUS and DR5::GFP expression but decreases the CYCB1;1::GUS expression in Arabidopsis root tips. Further studies indicated that LZR216 is able to inhibit the meristematic length and decrease the cell division capability but has little effect on the quiescent center function of the root meristem. Subsequently, it was also shown that LZR216 has no significant effects on the primary root length of the pin2 and aux1-7 mutants. Furthermore, LZR216 down-regulates the levels of PIN1-GFP, PIN2-GFP, PIN3-GFP, and AUX1-YFP. In addition, the wild-type Arabidopsis seedlings in the present of 1 or 5 µM NPA (an auxin transport inhibitor) were insensitive to LZR216-inhibited primary root elongation. Collectively, LZR216 regulates the development of root system architecture depending on polar auxin transport. This study shows a new insight on the ability of beneficial endophytic bacteria in regulating postembryonic root development.

     

Physiological and proteomic analysis of plant growth enhancement by the rhizobacteria <Emphasis Type="Italic">Bacillus</Emphasis> sp. JS

In this study, the effects of the plant growth-promoting rhizobacterium (PGPR), Bacillus sp. JS on the growth of tobacco (Nicotiana tabacum ‘Xanthi’) and lettuce (Lactuca sativa ‘Crispa’), were evaluated by comparing various growth parameters between plants treated with the bacterium and those exposed to water or nutrient broth as control. In both tobacco and lettuce, fresh weight and length of shoots were increased upon exposure to Bacillus sp. JS. To explain the overall de novo expression of plant proteins by bacterial volatiles, two-dimensional gel electrophoresis was performed on samples from PGPR-treated tobacco plants. Our results showed that chlorophyll a/b binding proteins were significantly up-regulated, and total chlorophyll content was also increased. Our findings indicate the potential benefits of using Bacillus sp. JS as a growth-promoting factor in agricultural practice, and highlight the need for further research to explore these benefits.     

Production of a Mixture of Fengycins with Surfactant and Antifungal Activities by <Emphasis Type="Italic">Bacillus</Emphasis> sp. MA04, a Versatile PGPR

Bacillus sp. strain MA04 a plant growth-promoting rhizobacteria (PGPR) showed hemolytic activity on blood agar plates, and the supernatant from liquid culture in nutrient broth at 24 h exhibited emulsification activity, suggesting the production of biosurfactants. In antagonist assays, the supernatant showed antifungal activity against phytopathogenic fungi such as Penicillium expansum, Fusarium stilboides, Sclerotium rolfsii y Rhizoctonia solani, finding a reduction of mycelial growth of all fungi tested, ranging from 35 to 69%, this activity was increased with time of culture, accomplishing percentages of inhibition up to 85% with supernatants obtained at 72 h. Then, the crude biorsurfactant (CB) was isolated from the supernatant in order to assay its antagonistic effect on the phytopathogens previously tested, finding an increase in the inhibition up to 97% at 500 mg/L of CB. The composition of CB was determined by infrared spectroscopy, identifying various functional groups related to lipopeptides, which were purified by high-performance liquid chromatography and analyzed by MALDI-TOF/TOF–MS, revealing a mixture of fengycins A and B whose high antifungal activity is been widely recognized. These results show that PGPR Bacillus sp. MA04 could also contribute to plant health status through the production of metabolites with antimicrobial activity.     

Plant growth-promoting potential of endophytic bacteria isolated from roots of wild <Emphasis Type="Italic">Dodonaea viscosa</Emphasis> L.

Dodonaea viscosa, a wild and perennial shrub that can tolerate harsh environmental conditions, was used for the isolation of its endophytic bacteria and their potential was explored for the promotion of Canola growth. The bacteria identified through 16S rRNA gene sequencing, belonged to ten different genera namely Inquilinus, Xanthomonas, Pseudomonas, Rhizobium, Brevundimonas, Microbacterium, Bacillus, Streptomyces, Agrococcus and Stenotrophomonas. All the strains produced small amount of IAA (indole acetic acid) in the absence of tryptophan and comparatively more in the presence of tryptophan. All the bacterial strains were positive for ammonia production, cellulase and pectinase activity, but few of them showed phosphate solubilization, siderophore and hydrogen cyanide production. Only three strains showed ACC (1-aminocyclopropane-1-carboxylate) deaminase activity when tested using in-vitro enzyme assay. Members of genera Bacillus, Pseudomonas and Streptomyces showed positive chitinase, protease and antifungal activity against two phytopathogenic fungi Aspergillus niger and Fusarium oxysoprum, while members of Xanthomonas, Pseudomonas and Bacillus showed significant root elongation of Canola which could be related with their positive plant-growth-promoting (PGP) traits. Among the three plant growth promoting Bacillus strains, B. idriensis is never reported before for its PGP activities. These results showed the potential of Dodonaea viscosa endophytic bacteria as PGPBs, which in future can be further explored for their host range/molecular mechanisms.     

Strains of <Emphasis Type="Italic">Bacillus</Emphasis> ssp. regulate wheat resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk.

The ability of Bacillus subtilis Cohn and Bacillus thuringiensis Berliner to induce systemic resistance in wheat plants to the casual agent of Septoria nodorum Berk., blotch has been studied. It has been shown that strains of Bacillus ssp. that possess the capacity for endophytic survival have antagonistic activity against this pathogen in vitro. A reduction of the degree of Septoria nodorum blotch development on wheat leaves under the influence of Bacillus spp. was accompanied by the suppression of catalase activity, an increase in peroxidase activity and H₂O₂ content, and expression of defence related genes such us PR-1, PR-6, and PR-9. It has been shown that B. subtilis 26 D induces expression levels of wheat pathogenesis-related (PR) genes which marks a SA-dependent pathway of sustainable development and that B. thuringiensis V-5689 and V-6066 induces a JA/ET-dependent pathway. These results suggest that these strain Bacillus spp. promotes the formation of wheat plant resistance to S. nodorum through systemic activation of the plant defense system. The designed bacterial consortium formed a complex biological response in wheat plants infected phytopathogen.     

Evaluation of the Diversity of Probiotic <Emphasis Type="Italic">Bacillus</Emphasis>, <Emphasis Type="Italic">Clostridium</Emphasis>, and <Emphasis Type="Italic">Bifidobacterium</Emphasis> Using the Illumina-Based Sequencing Method

Bacterial species of Bacillus, Lactobacillus, and Bifidobacterium in the intestinal tract have been used as probiotics. Selections for probiotic candidates by the culture-based approaches are time-consuming and labor-consuming. The aim of this study was to develop a new method based on sequencing strategies to select the probiotic Bacillus, Lactobacillus, and Bifidobacterium. The Illumina-based sequencing strategies with different specific primers for Bacillus, Clostridium, and Bifidobacterium were applied to analyze diversity of the genera in goat feces. The average number of different Bacillus, Clostridium, and Bifidobacterium OTUs (operational taxonomic units) at the 97% similarity level ranged from 1922 to 63172. The coverage index values of Bacillus, Clostridium, and Bifidobacterium calculated from the bacterial OTUs were 0.89, 0.99, and 1.00, respectively. The most genera of Bacillus (37.9%), Clostridium (53%), and Bifidobacterium (99%) were detected in goat feces by the Illumina-based sequencing with the specific primers of the genera, respectively. Higher phylogenetic resolutions of the genera in goat feces were successfully established. The results suggest that the selection for probiotic Bacillus, Clostridium, and Bifidobacterium based on the Illumina sequencing with their specific primers is reliable and feasible, and the core Bacillus, Clostridium, and Bifidobacterium species of healthy goats possess the potentials as probiotic microbial consortia.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Aseptic rearing procedure for the stinkbug <Emphasis Type="Italic">Plautia stali</Emphasis> (Hemiptera: Pentatomidae) by sterilizing food-derived bacterial contaminants

The stinkbug Plautia stali Scott is a notorious agricultural pest whose posterior midgut hosts specific bacteria essential for its growth and survival, highlighted as an experimental model for symbiosis studies. Some symbiotic bacteria of P. stali are cultivable, found free-living in and acquired from the environment, and, furthermore, some free-living environmental bacteria are potentially capable of establishing symbiotic association with P. stali. In this context, it is expected that such environmental bacteria may occasionally contaminate and infect the experimental insects maintained in the laboratory, which could potentially affect the functional analyses of the symbiosis. Here we report that such contamination events do occur under a laboratory rearing conditions for P. stali. When symbiont-deprived newborn nymphs from surface-sterilized eggs were reared in sterilized plastic containers with autoclaved water, most of them died as nymphs presumably as a result of aposymbiosis, but only a small fraction could attain adulthood and the adult insects were all infected with γ-proteobacteria allied to Pantoea and Enterobacter. A variety of bacteria, mainly Bacillus and also Pantoea and Enterobacter, were detected from peanuts and soybeans provided as food for P. stali. Autoclaving of peanuts and soybeans eradicated these bacteria but negatively affected the host survival, whereas ethanol sterilization of peanuts and soybeans removed Pantoea and Enterobacter, but not Bacillus, without negative effects on the host survival. On the basis of these results, we established a practical procedure for aseptic rearing of P. stali, which will enable reliable and strict analyses of host–symbiont interactions in the model symbiotic system.     

Diversity of endophytic fungal and bacterial communities in <Emphasis Type="Italic">Ilex paraguariensis</Emphasis> grown under field conditions

The composition and diversity of the endophytic community associated with yerba mate (Ilex paraguariensis) was investigated using culture-depending methods. Fungi were identified based on their micromorphological characteristics and internal transcribed spacer rDNA sequence analysis; for bacteria 16S rDNA sequence analysis was used. Fungal and bacterial diversity did not show significant differences between organ age. The highest fungal diversity was registered during fall season and the lowest in winter. Bacterial diversity was higher in stems and increased from summer to winter, in contrast with leaves, which decreased. The most frequently isolated fungus was Fusarium, followed by Colletotrichum; they were both present in all the sampling seasons and organ types assayed. Actinobacteria represented 57.5 % of all bacterial isolates. The most dominant bacterial taxa were Curtobacterium and Microbacterium. Other bacteria frequently found were Methylobacterium, Sphingomonas, Herbiconiux and Bacillus. Nitrogen fixation and phosphate solubilization activity, ACC deaminase production and antagonism against plant fungal pathogens were assayed in endophytic bacterial strains. In the case of fungi, strains of Trichoderma, Penicillium and Aspergillus were assayed for antagonism against pathogenic Fusarium sp. All microbial isolates assayed showed at least one growth promoting activity. Strains of Bacillus, Pantoea, Curtobacterium, Methylobacterium, Brevundimonas and Paenibacillus had at least two growth-promoting activities, and Bacillus, Paenibacillus and the three endophytic fungi showed high antagonistic activity against Fusarium sp. In this work we have made a wide study of the culturable endophytic community within yerba mate plants and found that several microbial isolates could be considered as potential inoculants useful for improving yerba mate production.     

Molecular diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing PGPR from wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) rhizosphere

Aims

The present study was planned to investigate the diversity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase producing bacteria from the rhizosphere of wheat plants and subsequent evaluation of selected PGPR on growth enhancement of wheat seedlings under drought and saline conditions.

Methods

ACC deaminase producing plant growth promoting rhizobacteria (PGPR) were isolated from the rhizosphere of wheat and identified using 16S rRNA gene sequence analysis. Isolates were evaluated for various direct and indirect plant growth promoting (PGP) traits. Plant inoculation experiment was conducted using isolates IG 19 and IG 22 in wheat to assess their plant growth promotion potential under salinity and drought stress.

Results

Thirty-eight ACC deaminase producing PGPR were isolated which belonged to 12 distinct genera and falling into four phyla γ-proteobacteria, β-proteobacteria, Flavobacteria and Firmicutes. Klebsiella sp. was the most abundant genera and followed by Enterobacter sp. The isolates exhibited ACC deaminase activities ranging from 0.106–0.980 μM α- ketobutyrate μg protein^?1 h^?1. The isolates showed multiple PGP traits such as IAA production, phosphate, zinc, potassium solubilization and siderophore production. Enterobacter cloacae (IG 19) and Citrobacter sp. (IG 22) inoculated wheat seedlings showed notable increases in fresh and dry biomass under non-stress as well as under stressed condition.

Conclusion

To the best of our knowledge this is the first report of presence of ACC deaminase activity and other PGP traits from the genus Citrobacter and Empedobacter. Our finding revealed that the γ-proteobacteria group dominated the wheat rhizosphere. Plant inoculation with PGPR could be a sustainable approach to alleviate abiotic stresses in wheat plants. These native PGPR isolates could be used as potential biofertilizers for sustainable agriculture.

     

Diversity of indigenous endophytic bacteria associated with the roots of Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis> L.) cultivars and their antagonism towards pathogens

The study aimed to reveal the diversity of endophytic bacteria in the roots of Chinese cabbage (CC) cultivated in two areas in Korea, namely, Seosang-gun (SS) and Haenam-gun (HN), and also in a transgenic plant (TP) from the laboratory. A total of 653 colonies were isolated from the interior of CC roots, comprising 118, 302, and 233 isolates from SS, HN, and TP samples, respectively. Based on 16S rRNA gene sequence analysis, the isolates belonged to four major phylogenetic groups: high-G+C Gram-positive bacteria (HGC-GPB), low-G+C Gram-positive bacteria (LGC-GPB), Proteobacteria, and Bacteriodetes. The most dominant groups in the roots of the SS, HN, and TP cultivars were LGC-GPB (48.3%), Proteobacteria (50.2%), and HGC-GPB (38.2%), respectively. Importantly, most of the isolates that produced cell-walldegrading enzymes belonged to the genus Bacillus. Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, TPR07), and Bacillus subtilis (TPR03) showed high antagonism against the tested food-borne pathogenic bacteria. In addition, Bacillus sp. (HNR03, TPR06), Bacillus pumilus (SSR07, HNR11, HNR17, TPR11), Microbacterium oxidans (SSR09, TPR04), Bacillus cereus HNR10, Pseudomonas sp. HNR13, and Bacillus subtilis (TPR02, TPR03) showed strong antagonistic activity against the fungi Phythium ultimum, Phytophthora capsici, Fusarium oxysporum, and Rhizoctonia solani. The endophytes isolated from the TP cultivar showed the strongest antagonistic reactions against pathogens. This study is the first report on endophytic bacteria from Chinese cabbage roots.     

Gram-Positive Bacteria with Probiotic Potential for the <Emphasis Type="Italic">Apis mellifera</Emphasis> L. Honey Bee: The Experience in the Northwest of Argentina

Apis mellifera L. is one of the most important natural pollinators of significant crops and flowers around the world. It can be affected by different types of illnesses: american foulbrood, nosemosis, varroasis, viruses, among others. Such infections mainly cause a reduction in honey production and in extreme situations, the death of the colony. Argentina is the world’s second largest honey exporter and the third largest honey producer, after China and Turkey. Given both the prominence of the honey bee in nature and the economic importance of apiculture in Argentina and the world, it is crucial to develop efficient and sustainable strategies to control honey bee diseases and to improve bee colony health. Gram-positive bacteria, such as lactic acid bacteria, mainly Lactobacillus, and Bacillus spp. are promising options. In the Northwest of Argentina, several Lactobacillus and Bacillus strains from the honey bee gut and honey were isolated by our research group and characterized by using in vitro tests. Two strains were selected because of their potential probiotic properties: Lactobacillus johnsonii CRL1647 and Bacillus subtilis subsp. subtilis Mori2. Under independent trials with both experimental and commercial hives, it was determined that each strain was able to elicit probiotic effects on bee colonies reared in the northwestern region of Argentina. One result was the increase in egg-laying by the queen which therefore produced an increase in bee number and, consequently, a higher honey yield. Moreover, the beneficial bacteria reduced the incidence of two important bee diseases: nosemosis and varroosis. These results are promising and extend the horizon of probiotic bacteria to the insect world, serving beekeepers worldwide as a natural tool that they can administer as is, or combine with other disease-controlling methods.     

Characterization of <Emphasis Type="Italic">lpaH2</Emphasis> gene corresponding to lipopeptide synthesis in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> HAB-2

Background

Bacillus spp. have prominent ability to suppress plant pathogens and corresponding diseases. Previous analyses of Bacillus spp. revealed numerous gene clusters involved in nonribosomal synthesis of cyclic lipopeptides with distinct antimicrobial action. The 4′-phosphopantetheinyl transferase (PPTase) encoded by sfp gene is a key factor in lipopeptide synthesis in Bacillus spp. In previous study, B. amyloliquefaciens strain HAB-2 was found to inhibit a broad range of plant pathogens, which was attributed to its secondary metabolite lipopeptide.

Results

A sfp homologue lpaH2 which encoded phosphopantetheinyl transferase but shared 71% sequence similarity was detected in strain HAB-2. Disruption of lpaH2 gene resulted in losing the ability of strain HAB-2 to produce lipopeptide, as well as antifungal and hemolytic activities. When lpaH2 replaced sfp gene of B. subtilis strain 168, a non-lipopeptide producer, the genetically engineered strain 168 could produced lipopeptides and recovered antifungal activity. Quantitative PCR assays indicated that, the expression level of lpaH2 in B. subtilis 168 strain decrease to 0.27-fold compared that of the wild type B. amyloliquefaciens strain HAB-2.

Conclusion

Few studies have reported about lpa gene which can replace sfp gene in the different species. Taken together, our study showed for the first time that lpaH2 from B. amyloliquefaciens could replace sfp gene.

     

The Mycotoxin Zearalenone Hinders <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation and Hyphal Morphogenesis

Yeast–mold mycobiota inhabit several natural ecosystems, in which symbiotic relationships drive strategic pathoadaptation. Mycotoxins are metabolites produced by diverse mycotoxigenic fungi as a defense against yeasts, though at times yeasts secrete enzymes that degrade, detoxify, or bio-transform mycotoxins. The present study is focused on the in vitro inhibitory effects of zearalenone (ZEN), a F2 mycotoxin produced by several Fusarium and Gibberella species, on different microbial strains. ZEN exhibited no effect on the planktonic growth or biofilms of several Gram positive and negative bacteria at the tested concentrations. Remarkably, Candida albicans biofilm formation and hyphal morphogenesis were significantly inhibited when treated with 100 µg/mL of ZEN. Likewise, ZEN proficiently disrupted pre-formed C. albicans biofilms without disturbing planktonic cells. Furthermore, these inhibitions were confirmed by crystal violet staining and XTT reduction assays and by confocal and scanning electron microscopy. In an in vivo model, ZEN significantly suppressed C. albicans infection in the nematode Caenorhabditis elegans. The study reports the in vitro antibiofilm efficacy of ZEN against C. albicans strains, and suggests mycotoxigenic fungi participate in asymmetric competitive interactions, such as, amensalism or antibiosis, rather than commensal interactions with C. albicans, whereby mycotoxins secreted by fungi destroy C. albicans biofilms.     

Taxonomic composition of bacteria associated with cultivated mollusks <Emphasis Type="Italic">Crassostrea lugubris</Emphasis> and <Emphasis Type="Italic">Perna viridis</Emphasis> and with the water of the Gulf of Nha Trang lagoon,Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

Isolation,characterization and virulence of bacteria from <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis> (Lepidoptera: Pyralidae)

Isolation, characterization and virulence of the culturable bacteria from entire tissues of larval Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) were studied to obtain new microbes for biological control. A total of 16 bacteria were isolated from living and dead larvae collected from different maize fields in the Eastern Black Sea Region of Turkey. The bacterial microbiota of O. nubilalis were identified as Pseudomonas aeruginosa (On1), Brevundimonas aurantiaca (On2), Chryseobacterium formosense (On3), Acinetobacter sp. (On4), Microbacterium thalassium (On5), Bacillus megaterium (On6), Serratia sp. (On7), Ochrobactrum sp. (On8), Variovorax paradoxus (On9), Corynebacterium glutamicum (On10), Paenibacillus sp. (On11), Alcaligenes faecalis (On12), Microbacterium testaceum (On13), Leucobacter sp. (On14), Leucobacter sp. (On15) and Serratia marcescens (On16) based on their morphological and biochemical characteristics. A partial sequence of the 16S rRNA gene was also determined to confirm strain identification. The highest insecticidal activities were obtained from P. aeruginosa On1 (80%), Serratia sp. On7 (60%), V. paradoxus On9 (50%) and S. marcescens On16 (50%) against larvae 14 days after treatment (p < 0.05). Also, the highest activity from previously isolated Bacillus species was observed from Bacillus thuringiensis subsp. tenebrionis Xd3 with 80% mortality within the same period (p < 0.05). Our results indicate that P. aeruginosa On1, Serratia sp. On7, V. paradoxus On9, S. marcescens On16 and B. thuringiensis subsp. tenebrionis Xd3 show potential for biocontrol of O. nubilalis.