Root colonization and growth enhancement in wheat and tomato by rhizobacteria isolated from the rhizoplane of grasses

Rhizobacteria isolated from the rhizoplane of grasses growing at the Nylsvlei Nature Reserve in South Africa were investigated for growth promotion and root colonization in wheat (Triticum aestivum L.) and tomato (Lycopersicon esculentum Mill.) under greenhouse and microplot field conditions. The identities of the isolates were determined by means of 16S rRNA gene sequencing as Bacillus simplex (KBS1F-3), Bacillus megaterium (NAS7-L), Bacillus cereus (KFP9-F) and Paenibacillus alvei (NAS6G-6). The three Bacillus strains were isolated from the perennial grass Themeda triandra while the Paenibacillus strain was isolated from another perennial grass Sporobolus fimbriatus. Enhanced plant shoot and root weight in wheat was achieved by single inoculation with three of the isolates whereas no significant increase was observed in root length. Combined inoculation of Paenibacillus alvei (NAS6G-6) and Bacillus cereus (KFP9-F) on wheat resulted in significant increase in these parameters. Single inoculations of Bacillus simplex (KBS1F-3) and Bacillus cereus (KFP9-F) resulted in significant increase in root and shoots fresh weight, root dry weight and total root length in tomatoes. Indoleacetic acid production, phosphate solubilization and siderophore secretion were studied as possible mechanisms by which the bacterial isolates enhanced plant growth. Root colonization was studied by means of spontaneous rifampicin resistant strains of the wild type isolates. Except for B. megaterium (NAS7-L), the rest of the isolates colonized the roots efficiently resulting in concentrations of 10⁶–10⁸ cfu g⁻¹ root. The root colonization of Bacillus simplex (KBS1F-3) and Paenibacillus alvei (NAS6G-6) was visualized by confocal scanning laser microscope (CSLM) after successful transformation of the isolates with the pNF8 plasmid carrying the gene for the green fluorescent protein (gfp).     

Survival and colonization of nematodes in a composting process

Nematodes are omnipresent in composts and are active in virtually all stages of the composting process. Major shifts in species composition, life strategies, and feeding behavior occur during the composting process. Due to the heat peak, nematodes can be virtually absent, but several taxa appear immediately when the temperatures drop. These comprise both taxa present before the heat peak and new taxa. However, it is not known how nematodes populate the compost. In this study, we aimed to assess the survival and colonization capacity of nematodes in compost. Our results showed that composting processes inaccessible to insects or not in contact with soil did not significantly influence nematode succession during composting. However, differences between treatments were found for some specific taxa (i.e., for Acrostichus sp., Neodiplogasteridae sp., Nygolaimoides sp., and Rhabditidae sp. 1), illustrating the importance of insects for the dispersal of nematodes to compost. Experiments in the lab with the blue bottle fly as a possible carrier demonstrated actual transport of nematodes isolated from compost by the fly (i.e., Halicephalobus cfr. gingivalis, Diploscapter coronatus, Diplogasteritus sp., Acrostichus sp., and Mesorhabditis sp.). Juveniles and dauer stages of Aphelenchoides sp., Panagrolaimus sp., and rhabditids survived an experimentally induced temperature peak, while members of Tylenchidae did not. In conclusion, our results indicate that the rapidly changing nematode community in compost is the result of both differential survival and colonization capacities.     

Transgenic tomato plants alter quorum sensing in plant growth-promoting rhizobacteria

Two Gram-negative, plant growth-promoting rhizobacteria (PGPRs), denominated as M12 and M14, were classified by 16S rDNA sequencing as Burkholderia graminis species. Both strains were shown to produce a variety of N-acyl-homoserine lactone (AHL) quorum sensing (QS) signalling molecules. The involvement of these molecules in plant growth promotion and the induction of protection against salt stress was examined. AHL production was evaluated in vitro by thin-layer chromatography using AHL biosensors, and the identity of the AHLs produced was determined by liquid chromatography-tandem mass spectrometry. The in situ production of AHLs by M12 and M14 in the rhizosphere of Arabidopsis thaliana plants was detected by co-inoculation with green fluorescent protein-based biosensor strains and confocal laser scanning microscopy. To determine whether plant growth promotion and protection against salt stress were mediated by QS, these PGPRs were assayed on wild-type tomato plants, as well as their corresponding transgenics expressing YenI (short-chain AHL producers) and LasI (long-chain AHL producers). In wild-type tomato plants, only M12 promoted plant growth, and this effect disappeared in both transgenic lines. In contrast, M14 did not promote growth in wild-type tomatoes, but did so in the LasI transgenic line. Resistance to salt stress was induced by M14 in wild-type tomato, but this effect disappeared in both transgenic lines. The strain M12, however, did not induce salt resistance in wild-type tomato, but did so in LasI tomato plants. These results reveal that AHL QS signalling molecules mediate the ability of both PGPR strains M12 and M14 to promote plant growth and to induce protection against salt stress.     

Diversity of cultivable rhizobacteria across tomato growing regions of Karnataka

Seven hundred and fifty-two rhizobacteria were isolated from 186 rhizosphere soil samples collected across tomato growing regions of Karnataka. Among them, 26% strains were Gram positive and other 74% were Gram negative and dominant being Bacillus and Pseudomonas. Sampling of different locations showed variation in species richness and diversity indices. Similarity matrix computed with Jaccard’s coefficient and principle coordinate analysis to correlate bacterial diversity revealed that rhizobacterial genera of Mysore, Mandya and Kolar soil samples were very closely related and rarefaction curve analysis indicated that these soil samples also harbored higher number of rhizobacteria which included all the genera studied. PGPR trait analysis revealed that most of the rhizobacteria were endowed with more than one beneficial trait which may act individually or simultaneously, and indole acetic acid production and phosphate solubilization are the two predominant traits exhibited by these rhizobacteria. Rhizobacterial isolates also showed a varied level of plant growth promotion traits and offered protection against fungal origin foliar and root pathogens. Among the nine regions studied, Mysore, Mandya and Kolar regions recorded higher percentage of promising PGPRs in comparison with other regions studied of Karnataka.     

Effect of plant growth promoting rhizobacteria on bacterial canker of tomato

Use of plant growth promoting rhizobacteria in managing bacterial canker disease of tomato was studied in the present work. Tomato seeds were treated with PGPR strains viz., Bacillus pumilus INR7, Bacillus pumilus SE34, Bacillus pumilus T4, Bacillus subtilis GBO3, Bacillus amyloliquefaciens IN937a and Brevibacillus brevis IPC11 were subjected for seed germination and seedling vigor. Among the PGPR strains tested, only three strains (IN937a, GBO3 and IPC11) which showed enhancement in the seed quality parameters like seed germination and seedling vigor, were further subjected for estimation of one of the defence-related enzymes, Phenylalanine Ammonia Lyase (PAL) with total phenol contents. The same three strains were recorded for maximum disease protection under greenhouse conditions. The level of PAL and total phenol contents increased significantly upon the PGPR treatment. The rate of reduction in the bacterial canker disease incidence was directly proportional to the amount of increased level of PAL and total phenol content. The possible uses of these PGPR strains in effective management of bacterial canker of tomato were discussed in the present work.     

Biocontrol of tomato late blight with the combination of epiphytic antagonists and rhizobacteria

Control of tomato late blight (LB) in Brazil is heavily based on chemicals. However, reduction in fungicide usage is required in both conventional and organic production systems. Assuming that biological control is an alternative for LB management, 208 epiphytic microorganisms and 23 rhizobacteria (RB) were isolated from conventional and organically grown tomato plants and tested for antagonistic activity against Phytophthora infestans. Based on in vitro inhibition of sporangia germination and detached leaflet bioassays, four EP microorganisms (Aspergillus sp., Cellulomonas flavigena, Candida sp., and Cryptococcus sp.) were selected. These microorganisms were applied either singly or combined on tomato plants treated or not with the RB Bacillus cereus. On control plants, LB progress rate (r), area under disease progress curve, and final disease severity were high. Lowest values of final severity were recorded on plants colonized by B. cereus and treated with C. flavigena, Candida sp. and Cryptococcus sp. There was no reduction on disease severity in plants treated only with RB. Biological control of LB resulted in low values of r and final severity. Integration of biological control with fungicides, cultural practices, and other measures can contribute to manage LB on tomato production systems.     

Induction of resistance in tomato against cucumber mosaic cucumovirus by plant growth-promoting rhizobacteria

Studies were done to evaluate specific strains of plant growth promoting rhizobacteria (PGPR) for induced resistance against cucumber mosaic cucumovirus(CMV) in tomato. In greenhouse experiments where plants were challenged by mechanical inoculation of CMV, the percentage of symptomatic plants in the most effective PGPR treatments ranged from 32 to 58%,compared with 88 to 98% in the nonbacterized, challenged disease control treatment. Field experiments were conducted in 1996 and 1997 to evaluate 4 PGPR strain treatments based on superior performance in the greenhouse studies. In the 1996field experiment, tomato plants treated with 3 PGPR strains exhibited a significantly lower incidence of CMV infection and significantly higher yields, compared with nonbacterized, CMV-challenged controls. In 1997, the overall percentages of plants infected with CMV in the control and PGPR treatments was higher than in 1996. CMV symptom development was significantly reduced on PGPR-treated plants in 1997compared with the control, but the percentage of infected plants and tomato yields were not significantly different among treatments. These results suggest that PGPR-mediated induced resistance against CMV infection following mechanical inoculation onto tomato can be maintained under field conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and characterization of phosphate solubilizing rhizobacteria to improve plant health of tomato

In the present study, 43 isolates of Phosphate solubilizing rhizobacteria (PSRB) were isolated from 37 rhizospheric soil samples of tomato collected from tomato growing regions of Karnataka. Among the 43 isolates, 33 were found to be positive for solubilizing both inorganic and organic forms of phosphorous. The isolates were analyzed for their ability to colonize roots of tomato and to increase the seed quality parameters under laboratory conditions. On the basis of above criteria, 16 isolates were selected for further studies. Organic acids from PSRB isolates were analyzed and phytase zymogram for two isolates viz., PSRB21 and 31 was prepared. Under greenhouse conditions, all selected isolates showed increased shoot length, root length, fresh weight, dry weight and phosphorous content of tomato seedlings to various extent with respect to control. Analysis of pH and available phosphorous in rhizosphere soil samples of 30 day-old-seedlings revealed that the available phosphorous content was high in rhizospheric soil samples of plants raised from seeds bacterized with PSRB isolates over control. Even though all selected PSRB’s were able to increase the plant growth, only few of them showed protection against fusarium wilt and none of them against early blight.     

Host-plant selectivity of rhizobacteria in a crop/weed model system

Belowground microorganisms are known to influence plants' performance by altering the soil environment. Plant pathogens such as cyanide-producing strains of the rhizobacterium Pseudomonas may show strong host-plant selectivity. We analyzed interactions between different host plants and Pseudomonas strains and tested if these can be linked to the cyanide sensitivity of host plants, the cyanide production of bacterial strains or the plant identity from which strains had been isolated. Eight strains (four cyanide producing) were isolated from roots of four weed species and then re-inoculated on the four weed and two additional crop species. Bacterial strain composition varied strongly among the four weed species. Although all six plant species showed different reductions in root growth when cyanide was artificially applied to seedlings, they were generally not negatively affected by inoculation with cyanide-producing bacterial strains. We found a highly significant plant species x bacterial strain interaction. Partitioning this interaction into contrasts showed that it was entirely due to a strongly negative effect of a bacterial strain (Pseudomonas kilonensis/brassicacearum, isolated from Galium mollugo) on Echinochloa crus-galli. This exotic weed may not have become adapted to the bacterial strain isolated from a native weed. Our findings suggest that host-specific rhizobacteria hold some promise as biological weed-control agents.     

Diatom colonization dynamics in a lotic system

A series of three overlapping sets of slides were exposed in riffle and pool habitats in a fourth order river in the Upper Peninsula of Michigan. Each set was exposed for 6–8 weeks and overlapped the preceeding set by 2 weeks. Diatom cell densities and community structure were determined after daily or weekly exposure periods for each set. The first set was placed on August 4, 1982, and the third set removed on October 16, 1982. Species diversity and evenness peaked quickly during colonization. Both indices decreased as the length of exposure increased. Early colonizing diatom species that occasionally accounted for large proportions of the total diatom community were soon replaced by other diatom species that tended to persist through time. Major dominant species were well established by day 28. Severe net cell losses (up to 17% of the total density) were recorded after only an 8–9 day exposure in both pools and riffles. Pool slides showed greater cell densities during the first few day's exposure than did slides exposed in riffle zones. After this brief conditioning period, however, the riffle slides showed more rapid cell growth and/or accumulation rates. Mean cell densities were similar between pool and riffle slides after 6–8 week exposures.Seasonal changes appeared to strongly influence diatom species succession. Seasonal changes in water velocity, temperature, or light may have the same effect as the more dramatic flood events which reset periphyton to earlier successional stages, resulting in increased major changes in species composition of the periphyton diatom community.     

Internal colonization of Salmonella enterica serovar Typhimurium in tomato plants

Several Salmonella enterica outbreaks have been traced back to contaminated tomatoes. In this study, the internalization of S. enterica Typhimurium via tomato leaves was investigated as affected by surfactants and bacterial rdar morphotype, which was reported to be important for the environmental persistence and attachment of Salmonella to plants. Surfactants, especially Silwet L-77, promoted ingress and survival of S. enterica Typhimurium in tomato leaves. In each of two experiments, 84 tomato plants were inoculated two to four times before fruiting with GFP-labeled S. enterica Typhimurium strain MAE110 (with rdar morphotype) or MAE119 (without rdar). For each inoculation, single leaflets were dipped in 10(9) CFU/ml Salmonella suspension with Silwet L-77. Inoculated and adjacent leaflets were tested for Salmonella survival for 3 weeks after each inoculation. The surface and pulp of ripe fruits produced on these plants were also examined for Salmonella. Populations of both Salmonella strains in inoculated leaflets decreased during 2 weeks after inoculation but remained unchanged (at about 10(4) CFU/g) in week 3. Populations of MAE110 were significantly higher (P<0.05) than those of MAE119 from day 3 after inoculation. In the first year, nine fruits collected from one of the 42 MAE119 inoculated plants were positive for S. enterica Typhimurium. In the second year, Salmonella was detected in adjacent non-inoculated leaves of eight tomato plants (five inoculated with strain MAE110). The pulp of 12 fruits from two plants inoculated with MAE110 was Salmonella positive (about 10(6) CFU/g). Internalization was confirmed by fluorescence and confocal laser microscopy. For the first time, convincing evidence is presented that S. enterica can move inside tomato plants grown in natural field soil and colonize fruits at high levels without inducing any symptoms, except for a slight reduction in plant growth.     

Transgenic expression of pear PGIP in tomato limits fungal colonization

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.     

Induced systemic resistance to tomato leaf curl virus and increased yield in tomato by plant growth promoting rhizobacteria under field conditions

The talc-based formulation of two Pseudomonas fluorescens strains (Pf1 and VPT10) and its mixture (with and without chitin) were tested against tomato leaf curl virus in tomato under greenhouse and field conditions. The mean percentage of tomato leaf curl virus infected plants were significantly lower (25%) with less symptom severity and delayed symptom expression up to nine additional days in Pseudomonas with chitin (VPT10 + chitin) treated tomato plants compared to non-bacterised control plants upon challenge inoculation with tomato leaf curl virus. Tomato leaf curl virus was partially purified and antiserum was developed. Using the antiserum the tomato leaf curl virus was detected in symptomatic leaves and in whitefly vector through direct antigen coating enzyme linked immunosorbent assay which revealed the low virus titre in Pseudomonas treated plants (VPT10 + chitin) and insect vector compared to untreated tomato plants. The results indicate the potentiality of plant growth promoting rhizobacteria strains and talc-powder formulations in the effective management of this tomato leaf curl virus in tomato under field conditions.     

Potential host colonization by insect herbivores in a warmer climate: a transplant experiment

We conducted a transplant experiment to investigate the potential colonization of a plant species by insect herbivores under a warmer climate. Acacia falcata seeds collected from four latitudes, encompassing the current coastal range of the species (1150 km), were grown in the same soil type and climatic conditions in a glasshouse. Plants were then transplanted to two sites, 280 km north of A. falcata's current coastal range; the transplant sites were 1.2 and 5.5°C warmer than the northernmost and southernmost boundaries of the species' current range, respectively. We compared the structure and composition of the herbivorous Hemiptera and Coleoptera communities on the transplants (i) to that of A. falcata within its current distribution, (ii) to a closely related Acacia species (Acacia leptostachya) that naturally occurred at the transplant sites, and (iii) among the A. falcata transplants originating from seeds collected at different latitudes. Herbivory on A. falcata was also compared between the transplants and the current distribution, and among transplant originating from different latitudes. Thirty species of externally feeding herbivorous Coleoptera and Hemiptera were collected from the transplanted A. falcata over a period of 12 months following transplantation. Guild structure of this herbivore community (based on the proportion of species within each of seven groups based on taxonomy and feeding style) did not significantly differ between the transplants and that found on A. falcata within its natural range, but did differ between the transplants and A. leptostachya. Rates of herbivory did not significantly differ between the transplants and plants at sites within the natural range. There were no significant differences in herbivore species richness or overall rates of herbivory on the transplants originating from different latitudes. In conclusion, host plant identity was apparently more important than climate in influencing the structure of the colonizing herbivore community. If this result holds for other plant–herbivore systems, we might expect that under a warmer climate, broad patterns in insect community structure and rates of herbivory may remain similar to that at present, even though species composition may change substantially.     

Polyploidy in tomato roots as affected by arbuscular mycorrhizal colonization

Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.     

Survival of and wheat-root colonization by alginate encapsulated Herbaspirillum spp

The survival ofHerbaspirillum spp. cells added directly or encapsulated in alginate beads and colonization of wheat roots was evaluated in soil microcosms. Cells entrapped in alginate in the presence of JNFb-broth and introduced into unplanted non-sterile clay loamy and sandy soils survived better than cells added directly to the same soils after 50 d incubation. On amendment by JNFb broth and/or skim milk the entrapped cells survived better than those prepared in water. Encapsulated cells survived better in a heavier textured soil (clay-loamy) than in a lighter (sandy) soil. Wheat plants growing in microcosms inoculated with various bead types from day 0 to day 30 exhibited high levels of histosphere colonization, nitrogenase activity (in situ) measured by acetylene reduction assay, plant dry mass and total N content but no symptoms of mottled stripe disease were observed. Comparable results of growth criteria and nitrogenase activity, but relatively lower bacterial populations, were obtained with wheat grown for 45 d after the inoculant had been introduced into the soil with different bead types.     

ACC-deaminase and/or nitrogen-fixing rhizobacteria and growth response of tomato (Lycopersicon pimpinellfolium Mill.)

The study aimed to identify and select important plant growth-promoting rhizobacteria (PGPR) and examine the response of tomato growth upon inoculation. Inoculation with rhizobacterial isolates increased all the measured physical, chemical, and enzymatic growth parameters compared to control. However, the TAN1 isolate had the highest effect, and significantly (P < 0.05) increased the root length (8.25-fold), root fresh (8.36-fold) and dry (12.6-fold) weight, shoot length (6.92-fold), shoot fresh (7.18-fold) and dry (6.90-fold) weight, number of leaves (11.0-fold), chlorophyll a (6.25-fold), chlorophyll b (10.7-fold), carotenoid contents (8.80-fold), seedlings fresh (9.0-fold) and dry (8.71-fold) weight, plant macronutrient uptake, i.e. N (7.7- and 8.9-fold), P (10.5- and 11.4-fold), K (7.8- and 8.8-fold), Ca (12.7- and 8.2-fold), and Mg (12.6- and 9-fold) in shoot and root, plant micronutrient uptake, i.e. Zn (6.6-, 10.2-), Cu (9.3-, and 10.3-fold), Fe (7.7- and 10.7-fold), and Mn (4.7- and 5.7-fold) in shoot and root and plant antioxidant enzymes, i.e. glutathione S-transferase (10.7-fold), peroxidase (8.1-fold), and catalase (10.5-fold). Our results concluded that inoculation of agricultural crops with rhizobacteria is a very useful approach to increase the plant growth. The rhizobacteria having both 1-aminocyclopropane-1-carboxylate (ACC) deaminase and nitrogen-fixing activity are more effective than rhizobacteria possessing either ACC-deaminase or nitrogen-fixing activity alone for growth promotion of crops.     

Ralstonia solanacearum colonization of tomato roots infected by Meloidogyne incognita

Bacterial wilt, caused by Ralstonia solanacearum, is one of the most serious diseases of tomato (Solanum lycopersicum). Concomitant infection of R. solanacearum and root‐knot nematode Meloidogyne incognita increases the severity of bacterial wilt in tomato, but the role of this nematode in disease complexes involving bacterial pathogens is not completely elucidated. Although root wounding by root‐knot nematode infection seems to play an important role, it might not entirely explain the increased susceptibility of plants to R. solanacearum. In the present study, green fluorescent protein (GFP)‐labelled R. solanacearum distribution was observed in the root systems of the tomato cultivar Momotaro preinoculated with root‐knot nematode or mock‐inoculated with tap water. Fluorescence microscopy revealed that GFP‐labelled R. solanacearum mainly colonized root‐knot nematode galls, and little or no green fluorescence was observed in nematode‐uninfected roots. These results suggest that the gall induced by the nematode is a suitable location for the growth of R. solanacearum. Thus, it is crucial to control both R. solanacearum and root‐knot nematode in tomato production fields to reduce bacterial wilt disease incidence and effects.