The main food source of free-living nematodes in the soil environment is bacteria, which can affect nematode development, fecundity and survival. In order to occupy a reliable source of bacterial food, some nematodes have formed specific relationships with an array of invertebrate hosts (where bacteria proliferate once the hosts dies), thus forming a tritrophic system of nematode, bacteria and insect or other invertebrates. We isolated 768 Bacillus strains from soil (from Germany and the UK), horse dung and dung beetles and fed them to the genetically tractable free-living nematodes Caenorhabditis elegans and Pristionchus pacificus to isolate nematocidal strains. While C. elegans is a bacteriovorous soil nematode, P. pacificus is an omnivorous worm that is often found in association with scarab beetles. We found 20 Bacillus strains (consisting of B. cereus, B. weihenstephanensis, B. mycoides and Bacillus sp.) that were pathogenic to C. elegans and P. pacificus causing 70% to 100% mortality over 5 days and significantly affect development and brood size. The most pathogenic strains are three B. cereus-like strains isolated from dung beetles, which exhibit extreme virulence to C. elegans in less than 24 h, but P. pacificus remains resistant. C. elegans Bre mutants were also highly susceptible to the B. cereus-like strains indicating that their toxins use a different virulence mechanism than B. thuringiensis Cry 5B toxin. Also, mutations in the daf-2/daf-16 insulin signaling pathway do not rescue survival. We profiled the toxin genes (bcet, nhe complex, hbl complex, pcpl, sph, cytK, piplc, hly2, hly3, entFM and entS) of these three B. cereus-like strains and showed presence of most toxin genes but absence of the hbl complex. Taken together, this study shows that the majority of naturally isolated Bacillus from soil, horse dung and Geotrupes beetles are benign to both C. elegans and P. pacificus. Among 20 pathogenic strains with distinct virulence patterns against the two nematodes, we selected three B. cereus-like strains to investigate resistance and susceptibility immune responses in nematodes.

The biocontrol potential of Bacillus subtilis isolated from cow dung microflora was investigated in vitro and in vivo against two postharvest yam pathogenic fungi, Fusarium oxysporum and Botryodiplodia theobromae. B. subtilis strains inhibited the growth of F. oxysporum and B. theobromae in vitro in liquid medium in the range of 49.3-56.6% and in solid medium in the range of 31.0-36.0%, in comparison to the corresponding growth of fungi without bacterial inoculation. The interaction between B. subtilis CM1 and F. oxysporum was also studied by scanning electron microscopy. Chitinase production was demonstrated in vitro when B. subtilis was grown in the presence of colloidal chitin as the sole carbon source in a liquid medium. In vivo study showed that B. subtilis strains inhibited the growth of fungi (F. oxysporum and B. thobromae) up to 83% in wound cavities of yam tubers.

In extraction of starch from cassava (Manihot esculenta Crantz), one of the major solid waste released is fibrous residues which constitute 15-20% by weight of the cassava chips/tuber processed. Production of alpha -amylase under solid state fermentation by Bacillus subtilis CM3 has been investigated using cassava fibrous residue. Response surface methodology (RSM) was used to evaluate the effect of the main variables, i.e. incubation period, initial medium pH, moisture holding capacity and temperature on enzyme production. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha -amylase production. The experimental results showed that the optimum incubation period, initial medium pH, moisture holding capacity and temperature were 6 days, 8.0, 70% and 50 degrees C, respectively.

The biocontrol potential of Bacillus subtilis isolated from cow dung microflora was investigated in vitro and in vivo against two postharvest yam pathogenic fungi, Fusarium oxysporum and Botryodiplodia theobromae. B. subtilis strains inhibited the growth of F. oxysporum and B. theobromae in vitro in liquid medium in the range of 49.3-56.6% and in solid medium in the range of 31.0-36.0%, in comparison to the corresponding growth of fungi without bacterial inoculation. The interaction between B. subtilis CM1 and F. oxysporum was also studied by scanning electron microscopy. Chitinase production was demonstrated in vitro when B. subtilis was grown in the presence of colloidal chitin as the sole carbon source in a liquid medium. In vivo study showed that B. subtilis strains inhibited the growth of fungi (F. oxysporum and B. thobromae) up to 83% in wound cavities of yam tubers.

In extraction of starch from cassava (Manihot esculenta Crantz), one of the major solid waste released is fibrous residues which constitute 15-20% by weight of the cassava chips/tuber processed. Production of alpha -amylase under solid state fermentation by Bacillus subtilis CM3 has been investigated using cassava fibrous residue. Response surface methodology (RSM) was used to evaluate the effect of the main variables, i.e. incubation period, initial medium pH, moisture holding capacity and temperature on enzyme production. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha -amylase production. The experimental results showed that the optimum incubation period, initial medium pH, moisture holding capacity and temperature were 6 days, 8.0, 70% and 50 degrees C, respectively.

There is a growing interest to find alternate bioresources for production of ethanol, apart from cane/sugar beet molasses and starchy crops like sweet sorghum, cassava and sweet potato. Mahula (Madhuca latifolia L.) is a forest tree abundantly available in the Indian subcontinent and its flowers are very rich in fermentable sugars (28.1-36.3 g 100 g(-1)). Batch fermentation of fresh and 12-month-stored flowers with free (whole cells) and immobilized cells of Saccharomyces cerevisiae (strain CTCRI) was carried out in 2-l Erlenmeyer flasks. The ethanol yields were 193 and 148 g kg(-1)(using free cells) and 205 and 152 g kg(-1)(using immobilized cells) from fresh and 12-month-stored mahula flowers, respectively.

Sweetpotato storage roots are subjected to several forms of post harvest spoilage in the tropical climate during transportation from farmers' field to market and in storage. These are due to mechanical injury, weight loss, sprouting, and pests and diseases. Sweetpotato weevil is the single most important storage pest in tropical regions for which no control measures or resistant variety are yet available. Several microorganisms (mostly fungi) have been found to induce spoilage in stored sweetpotatoes. The most important among them are Botryodiplodia theobromae, Ceratocystis fimbriata, Fusarium spp., and Rhizopus oryzae. The other less frequently occurring spoilage microorganisms include Cochliobolus lunatus (Curvularia lunata), Macrophomina phaseolina, Sclerotium rolfsii, Rhizoctonia solani, Plenodomus destruens. Microbial spoilage of sweetpotato is found associated with decrease in starch, total sugar, organic acid (ascorbic acid and oxalic acid) contents with concomitant increase in polyphenols, ethylene, and in some instances phytoalexins. Several methods are used to control microbial spoilage. Curing to promote wound healing is found as the most suitable method to control microbial spoilage. Curing naturally occurs in tropical climates where mean day temperature during sweetpotato harvesting season (February-April) invariably remains at 32-35 degrees C and relative humidity at 80-95%. Sweetpotato varieties varied in their root dry matter content, and low root dry matter content attributed for their high curing efficiency. Curing efficiency of varieties also differed in response to curing periods. Fungicide treatment, bio-control, gamma irradiation, hydro warming, and storage in sand and saw dust were found to have intermediate impacts in controlling spoilage and enhancing shelf life of sweetpotato roots. Breeding program has to be chalked out to develop new varieties suitable to curing under tropical conditions in addition to developing varieties having multi-spectrum resistance to major post harvest rot pathogens and sweetpotato weevils.

The fungi Botryodiplodia theobromae and Rhizopus oryzae produce extracellular amylase when grown on a liquid medium containing 2%(WN) soluble starch or cassava starch residue(CSR)(as starch equivalent), a waste generated after extraction of starch from cassava, as the sole carbon source. Using CSR as the sole carbon source, the highest amylase activity of 3.25 and 3.8 units (mg, glucose released x ml(-1) x h(-1)) were obtained in shake flask cultures during the late stationary phase of growth of B. theobromae and R. oryzae, respectively. These values were slightly lower than the values obtained using soluble starch as the carbon source. Maximum enzyme synthesis in CSR incorporated medium occurred at the growth temperature of 30 degrees C and pH 6.0. Presence of inorganic NH4+ salts like ammonium acetate and ammonium nitrate in culture medium yielded more amylase than the other nitrogen sources. Amylase(s) production in the controlled environment of a Table-Top glass Jar Fermenter (2-L capacity) was 4.8 and 5.1 units for B. theobromae and R. oryzae, respectively using CSR as the carbon substrate. It is concluded that CSR, a cheap agricultural waste obtained after starch extraction from cassava could replace soluble starch as carbon substrate for commercial production of fungal amylase(s).

The availability of a large number of tRNA sequences has created a need for computer programs to store the data. A program written in FORTRAN language allows one to store tRNA sequences and to retrieve information. The data are stored in such a way that it allows a direct comparison of various tRNA sequences. Moreover, the program draws the 'cloverleaf' structure of any stored tRNA sequence and also provides the characteristics of minor, modified nucleosides and major references. Additions and corrections to the program and stored data can be made easily and an individual does not need computer programming skills to use the program.

Aims:  Our goal was to understand the symbiotic behaviour of a Mesorhizobium strain expressing an exogenous 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which was used as an inoculant of chickpea (Cicer arietinum) plants growing in soil. Methods and Results:  Mesorhizobium ciceri LMS-1 (pRKACC) was tested for its plant growth promotion abilities on two chickpea cultivars (ELMO and CHK3226) growing in nonsterilized soil that displayed biotic and abiotic constraints to plant growth. When compared to its wild-type form, the M. ciceri LMS-1 (pRKACC) strain showed an increased nodulation performance of c. 125 and 180% and increased nodule weight of c. 45 and 147% in chickpea cultivars ELMO and CHK3226, respectively. Mesorhizobium ciceri LMS-1 (pRKACC) was also able to augment the total biomass of both chickpea plant cultivars by c. 45% and to reduce chickpea root rot disease susceptibility. Conclusions:  The results obtained indicate that the production of ACC deaminase under free living conditions by Mesorhizobium strains increases the nodulation, plant growth abilities and biocontrol potential of these strains. Significance and Impact of the Study:  This is the first study regarding the use of a transformed rhizobial strain expressing an exogenous ACC deaminase in different plant cultivars growing in soil. Hence, obtaining Mesorhizobium strains with high ACC deaminase activity is a matter of extreme importance for the development of inoculants for field applications.

BACKGROUND Plant growth-promoting rhizobacteria (PGPR) can be potential agents for biological control of plant pathogens, while their combined use with conventional pesticides may increase their efficacy and broaden the disease control spectrum. The effect of four different Bacillus sp. PGPR strains (B. subtilis GB03 and FZB24, B. amyloliquefaciens IN937a and B. pumilus SE34) applied individually and in mixtures, as well as in combined use with acibezolar-S-methyl (ASM) and hymexazol, on plant growth promotion and on the control of Fusarium crown and root rot (FCRR) of tomato was evaluated. RESULTS All PGPR strains promoted the tested plant growth characteristics significantly. A higher promoting effect was provided by SE34. Experiments on population dynamics of PGPR strains revealed that, after 28 days of incubation, populations of strain SE34 remained stable, while the remaining bacterial strains showed a slight decline in their population densities. The GB03 and FZB24 strains provided a higher disease suppression when applied individually. However, application of IN937a in a mixture with GB03 provided a higher control efficacy of Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Treatment of tomato plants with ASM resulted in a small reduction in disease index, while application of hymexazol provided significantly higher control efficacy. Combined applications of the four PGPR strains with either ASM or hymexazol were significantly more effective. CONCLUSION The results of the study indicate that, when bacilli PGPR strains were combined with pesticides, there was an increased suppression of Forl on tomato plants, and thus they may prove to be important components in FCRR integrated management.

Thirty endophytic bacteria were isolated from various plant species growing near Saint-Petersburg, Russia. Based on a screening for various traits, including plant-beneficial properties and DNA fragment patterns, potential siblings were removed. The remaining isolates were taxonomically identified using 16S rDNA sequences and potential human and plant pathogens were removed. The remaining strains were tested for their ability to promote radish root growth and to protect tomato plants against tomato foot and root rot. One strain, Bacillus subtilis HC8, isolated from the giant hogweed Heracleum sosnowskyi Manden, significantly promoted plant growth and protected tomato against tomato foot and root rot. Metabolites possibly responsible for these plant-beneficial properties were identified as the hormone gibberellin and (lipo)peptide antibiotics respectively. The antibiotic properties of strain HC8 are similar to those of the commercially available plant-beneficial strain Bacillus amyloliquefaciens FZB42. However, thin layer chromatography profiles of the two strains differ. It is speculated that endophytes such as B. subtilis HC8 contribute to the fast growth of giant hogweed.

AIMS To isolate and characterize phosphate-solubilizing strains from a constrained environment such as the Salado River Basin and to assess their phosphate-solubilizing mechanisms, to further selection of the most promising strains to inoculate and improve the implantation and persistence of Lotus tenuis in the most important area devoted to meat-cow production in Argentina. METHODS AND RESULTS Fifty isolates were obtained and through BOX-PCR analysis, 17 non-redundant strains were identified. Subsequently, they were found to be related to Pantoea, Erwinia, Pseudomonas, Rhizobium and Enterobacter genera, via 16S rRNA gene sequence analysis. This was in agreement with the clusters obtained by antibiotic resistance analysis. All isolates were tested for their phosphate-solubilizing activity and selected strains were inoculated onto L. tenuis plants. The most efficient isolate, was identified as Pantoea eucalypti, a novel species in terms of plant growth-promoting rhizobacteria. CONCLUSIONS The isolates obtained in this study showed a significant in vitro plant-growth promoting activity onto Lotus tenuis and the best of them solubilizes phosphate mainly via induction of the metabolism through secretion and oxidation of gluconic acid. SIGNIFICANCE AND IMPACT OF THE STUDY The use of these bacteria as bioinoculants, alone or in combination with nitrogen-fixing micro-organisms, could be a sustainable practice to facilitate the nutrient supply to Lotus tenuis plants and preventing negative side-effects such as eutrophication.

Dark septate endophytes (DSE) are common and abundant root-colonizing fungi in the native tallgrass prairie. To characterize DSE fungi were isolated from roots of mixed tallgrass prairie plant communities. Isolates were grouped according to morphology, and the grouping was refined by ITS-RFLP and/or sequencing of the ITS region. Sporulating species of Periconia, Fusarium, Microdochium and Aspergillus were isolated along with many sterile fungi. Leek resynthesis was used to quickly screen for DSE fungi among the isolates. Periconia macro-spinosa and Microdochium sp. formed typical DSE structures in the roots; Periconia produced melanized intracellular microsclerotia in host root cortex, whereas Microdochium produced abundant melanized inter- and intracellular chlamydospores. To further validate the results of the leek resynthesis growth responses of leek and a dominant prairie grass, Andropogon gerardii, were assessed in a laboratory resynthesis system. Leek growth mainly was unresponsive to the inoculation with Periconia or Microdochium, whereas Andropogon tended to respond positively. Select Periconia and Microdochium isolates were tested further for their enzymatic capabilities and for ability to use organic and inorganic nitrogen sources. These fungi tested positive for amylase, cellulase, polyphenol oxidases and gelatinase. Periconia isolates used both organic and inorganic nitrogen sources. Our study identified distinct endophytes in a tallgrass prairie ecosystem and indicated that these endo-phytes can use a variety of complex nutrient sources, suggesting facultative biotrophic and saprotrophic habits.

Soil samples, collected after the fire operations at agricultural sites under shifting cultivation in northeast India, were subjected to physico-chemical and microbial analysis. The fire affected various physico-chemical properties of the soil. Significant differences in pH and electrical conductivity were recorded in soil of fired and fallow plots. Significantly higher amounts of total organic carbon and nitrogen were estimated in fallow plots as compared to the fired. Difference in total phosphates was not significant. The fire operations resulted in stimulation of microbial communities. The bacteria were the most affected group followed by actinomycetes and fungi, respectively. The bacterial and actinomycetes counts were significantly higher in fired plots as compared to the fallow plots. The representative bacterial species recovered from the 'fired plots' belonged to the genus Bacillus and Pseudomonas. 16S rRNA analysis revealed their maximum similarity with B. clausii, B. licheniformis, B. megaterium, B. subtilis, B. thuringiensis, P. aeruginosa and P. stutzeri. Most of these species were found to be positive for phosphate solubilization and antagonism in plate based assays. In view of the importance of Bacillus and Pseudomonas species in plant growth promotion and biocontrol, recovery of these species after fire operations is indicative of the microbiological merit of shifting cultivation.

The production of indole-3-acetic acid (IAA), by rhizobacteria, has been associated with plant growth promotion, especially root initiation and elongation. Isolate TO3 selected from 103 fluorescent pseudomonads, identified as Pseudomonas aeruginosa, showed maximum production of IAA. Isolate TO3 having biocontrol activity against Macrophomina phaseolina also showed production of siderophore and HCN was used to screen the role of bacterial IAA in reducing the level of charcoal rot disease occurrence in chickpea. Four IAA defective stable mutants of isolate TO3 having biocontrol activity against M. phaseolina were developed through 5-bromouracil mutagenesis. Mutant TO(52) showed 76.47% reduction in production of IAA. Standard IAA was used in similar concentration as present in cell-free culture supernatant of wild isolate TO3 and its mutant TO(52). The in vitro and in vivo study showed that IAA-defective mutant TO(52) caused reduced biocontrol and plant growth promotory activity than wild isolate TO3. Standard IAA showed comparable biocontrol activity to the culture supernatant. To some extent better biocontrol and growth promotory activity in supernatant than standard IAA indicates the synergistic role of siderophore and HCN. The study clearly reports the role of bacterial IAA in suppression of charcoal rot disease of chickpea.

Abstract The plasmid pME6863, carrying the aiiA gene from the soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme able to degrade N-acyl-homoserine lactones (AHLs), was introduced into the rhizosphere isolate Pseudomonas fluorescens P3. This strain is not an effective biological control agent against plant pathogens. The transformant P. fluorescens P3/pME6863 acquired the ability to degrade AHLs. In planta, P. fluorescens P3/pME6863 significantly reduced potato soft rot caused by Erwinia carotovora and crown gall of tomato caused by Agrobacterium tumefaciens to a similar level as Bacillus sp. A24. Little or no disease reduction was observed for the wild-type strain P3 carrying the vector plasmid without aiiA. Suppression of potato soft rot was observed even when the AHL-degrading P. fluorescens P3/pME6863 was applied to tubers 2 days after the pathogen, indicating that biocontrol was not only preventive but also curative. When antagonists were applied individually with the bacterial plant pathogens, biocontrol activity of the AHL degraders was greater than that observed with several Pseudomonas 2,4-diacetylphloroglucinol-producing strains and with Pseudomonas chlororaphis PCL1391, which relies on production of phenazine antibiotic for disease suppression. Phenazine production by this well characterized biological control strain P. chlororaphis PCL1391 is regulated by AHL-mediated quorum sensing. When P. chlororaphis PCL1391 was co-inoculated with P. fluorescens P3/pME6863 in a strain mixture, the AHL degrader interfered with the normally excellent ability of the antibiotic producer to suppress tomato vascular wilt caused by Fusarium oxysporum f. sp. lycopersici. Our results demonstrate AHL degradation as a novel biocontrol mechanism, but also demonstrate the potential for non-target interactions that can interfere with the biocontrol efficacy of other strains.

Root rot caused by soil borne pathogenic fungi is the most sever disease attacks lupine plants. Isolation trials from diseased plants in some areas of Dakahlia Province (Egypt) was carried out. Rhizoctonia solani and Fusarium solani proved to be the most dominant isolates. Meanwhile, Fusarium oxysporum and Sclerotium rolfsii were less frequent. Efficacies of some plant resistance elicitors viz.: chitosan (CHI), Salicylic Acid (SA) and hydroquinone (HQ) in comparing to the fungicide Rhizolex T-50 as seed treatments showed significant reduction in the fungal growth in vitro. Chitosan at 8 g L(-1) and fungicide completely inhibited the growth of all isolated fungi, while SA at 1.4 g L(-1) and HQ at 1.2 g L(-1) inhibited the growth of Fusarium solani and F. oxysporum, respectively. The greenhouse experiments showed that S. rolfesii (No. 6) and R. solani (No. 2) followed by F. solani (No. 5) and F. oxysporum (No. 9) were the most aggressive root rot fungi. Soaking susceptible lupine seeds (Giza 1) in each one of the three selected elicitors showed a significant reduction in seedlings mortality. CHI at 8 g L(-1) was superior in increasing the percentage of healthy plants to record 72.5, 80.9, 62.7and 64.3%, when seeds were grown in soil infested with of F. solani, F. oxysporum, R. solani and S. rolfesii, respectively. These results were confirmed under field conditions in two different locations i.e., Tag El-Ezz and El-Serow Research Stations. CHI 8 g L(-1) proved to be the best elicitor after fungicide, in reducing lupine root rot disease. It showed 41 and 60% reduction in the plants mortality comparing to 56.37 and 69.13% in case of Rhizolex-T in Tag El-Ezz and El-Serow locations, respectively. The treatments were accompanied with a significant increase in lupine growth parameters, yield components and physiological aspects. Application of CHI at 8 g L(-1) or HQ at 1.2 g L(-1) was the most potent in this respect as compared to check treatment.

AIM To evaluate Ochrobactrum anthropi TRS-2 isolated from tea rhizosphere and its talc based formulation for growth promotion and management of brown root rot disease of tea. METHODS AND RESULTS Ochrobactrum anthropi TRS-2, isolated from tea rhizosphere could solubilize phosphate, produce siderophore and IAA in vitro and also exhibited antifungal activity against six test pathogens. Application of an aqueous suspension of O. anthropi to the rhizosphere of nursery grown tea seedlings of five varieties of tea (TV-18, T-17, HV-39, S-449, UP-3 and) led to enhanced growth of the treated plants, as evidenced by increase in height, in the number of shoots and number of leaves per shoot. Treatment with O. anthropi also decreased brown root rot of tea, caused by Phellinus noxius. Multifold increase in activities of chitinase, beta-1,3-glucanase, peroxidase and phenylalanine ammonia lyase in tea plants was observed on application of O. anthropi to soil followed by inoculation with P. noxius. A concomitant increase in accumulation of phenolics was also obtained. Further, talc based formulation of O. anthropi was prepared and its survival determined every month up to a period of 12 months. Ochrobactrum anthropi could survive in the formulation up to a period of 9 months with a concentration of 7.0 log(10) CFU g(-1), after which there was a decline. Talc formulation was as effective as aqueous suspensions in both plant growth promotion and disease suppression. CONCLUSION Ochrobactrum anthropi, either in aqueous suspension or as talc formulation induced growth of tea plants and suppressed brown root rot disease. It induced defense responses in tea plants. SIGNIFICANCE AND IMPACT OF THE STUDY Ochrobactrum anthropi and its talc based formulation can be considered as an addition to available plant growth promoting rhizobacteria (PGPR) currently being used for field application. The present study offers a scope of utilizing this bacterium for growth promotion and disease management which would help in reduction of the use of chemicals in tea plantations.