Survival of Agrobacterium radiobacter K84 on various carriers for crown gall control.

Screening was performed on nine carriers to find an improved formulation for Agrobacterium radiobacter K84 cells. The survival data showed that it is possible to preserve A. radiobacter cells on dry solid supports for a long time provided that the storage temperature is 4 degrees C and that the inoculation volume for 4 x 10(9) CFU g-1 is not less than 0.15 ml g of carrier-1. On the other hand, a substantial carrier water content was necessary for room temperature storage. Many materials proved to be suitable as microbial carriers; in some cases, vermiculite allowed long storage times comparable to those reported for peat or carboxymethyl cellulose, which are already employed in some commercial A. radiobacter K84 products. Furthermore, vermiculite assured full and immediate biological activity in the prevention of crown gall, showing that it is suitable for a new formulation of strain K84. A hypothesis to explain the different survival abilities in wet and dry conditions is presented.     

Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113

Sugarbeet seeds used by farmers are often pelleted using an EB^TM-based mix. During the pelleting process, the seeds are dried immediately after application of the mix. In this work, the effects of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113Rif were investigated using a 1:1 EB^TM/vermiculite mix and sugarbeet seeds pelleted with this material. Growing F113Rif for 3 d (28 °C) within the EB^TM/vermiculite mix amended with nutrients (sucrose asparagine broth), instead of adding the cells to the unamended mix immediately before drying the mix or the pelleted sugarbeet seeds, resulted in improved survival of the strain in the mix or on the seeds, respectively, during subsequent storage. A slower drying (20 h instead of 3 h) of the F113Rif-inoculated EB^TM/vermiculite mix to 11% w/w water content enhanced strain survival in the mix during storage, but the drying conditions studied had no effect on inoculant survival on the seed during storage when pelleted seeds were dried to 10% w/w water content. Biological control of damping-off disease of sugarbeet (caused by Pythium spp.) in soil microcosms was achieved when F113Rif was inoculated in the unamended mix 3 d before pelleting the seeds, but not when nutrient-amended mix was used. Inoculum preparation and drying of the formulation are key factors to consider when optimizing the use of a commercial EB^TM/vermiculite seed formulation for delivery of a biocontrol Pseudomonas inoculant.     

Fate of Agrobacterium radiobacter K84 in the environment.

Agrobacterium radiobacter K84 is an effective, commercially applied, biological control agent for the plant disease crown gall, yet little is known about the survival and dissemination of K84. To trace K84 in the environment, spontaneous antibiotic-resistant mutants were used. Growth rates and phenotypes of streptomycin- or rifampin-resistant K84 were similar to those of the parental K84, except the rifampin-resistant mutant produced less agrocin 84 as determined by bioassay. K84 and a strain of Agrobacterium tumefaciens established populations averaging 10(5) CFU/g in the rhizosphere of cherry and persisted on roots for 2 years. K84 established rhizosphere populations between 10(4) and 10(6) CFU/g on cherry, ryegrass, and 11 other herbaceous plants. Populations of K84 declined substantially in fallow soil or water over a 16-week period. K84 was detected in the rhizosphere of ryegrass located up to 40 cm from an inoculum source, indicating lateral dissemination of K84 in soil. In gall tissue on cherry, K84 established populations of 10(5) CFU/g, about 10- to 100-fold less than that of the pathogen. These data demonstrate that K84 persists for up to 2 years in a field environment as a rhizosphere inhabitant or in association with crown gall tissue.     

Survival of several <Emphasis Type="Italic">Rhizobium/Bradyrhizobium</Emphasis> strains on different inoculant formulations and inoculated seeds

The effect of a variety factors on the survival of several rhizobia strains on inoculants and inoculated seeds has been evaluated. Since the rhizobia strains showed different cell-density-evolution patterns on peat-based inoculants and on inoculated seeds, several inoculant formulations with highly effective Rhizobium/Bradyrhizobium strains (for Lupinus, Hedysarum, Phaseolus and Glycine max.) were monitored under the following storage conditions: (a) the inoculants were kept refrigerated (at 4 °C), or (b) at room temperature (25 °C). The effect of water content (30–50%, w/w) in the inoculants as well as that of several seed-coating adhesives were also investigated. Alternative carriers including perlite and vermiculite were tested. For all of the strains, survival on sterile peat-based inoculants was higher than on the corresponding unsterile peat formulation; for the latter, refrigerated storage conditions are recommended to ensure high bacterial densities. The water content of the inoculants had a differential effect on strain survival depending on the sterility of the peat, such that a high water content was more detrimental when unsterilized peat was employed. The best adherent for rhizobia survival was a gum arabic/water solution. Perlite was as effective as peat in maintaining a high population of rhizobia, at least for 6 months of storage. Electronic Publication     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

Attachment of Agrobacterium tumefaciens B6 and A. radiobacter K84 to Tomato Root Tips

Agrobacterium tumefaciens B6 and the avirulent Agrobacterium radiobacter strain K84 attached to in vitro-cultured tomato root tips, but the binding of strain B6 to root tips was greater than the binding of strain K84. Strain K84 was not able to block the attachment of A. tumefaciens B6 to in vitro-cultured tomato root tips.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach x almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produce an antibiotic‐like substance,active in vitro against A. tumefaciens and phytopathogenic Erwinia and Pseudomonas spp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr^‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr^‐.     

Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti

The inoculation of legumes with rhizobia is used to maximise nitrogen fixation and enhance the plant yield without using N fertilisers. For this reason many inoculant types were developed and optimised. In our study, the effects of the growth medium, the carrier, the temperature and the storage period were determined on the survival of Sinorhizobium meloliti. Secondary sludge from Communauté Urbaine de Quebec wastewater treatment plant and standard medium (YMB) were used for rhizobial growth. Dehydrated sludge from Jonquière wastewater treatment plant, peat and a mixture of peat and sludge were used as carrier materials. Results showed that the wastewater sludge offered better protection for rhizobia survival during freezing and thawing at -20 degrees C than the standard medium. In general, results also showed the suitability of using sludge as a carrier because it had the same or a higher potential than peat to support survival of S. meliloti. In the case of YMB-grown rhizobia, peat- and sludge-based carriers appeared to be similar in terms of survival rate during the storage at 4 and 25 degrees C. For secondary sludge-grown rhizobia, the survival was better in sludge than in peat based carrier. Generally, the cell count remained higher than 10(8) cells/g for up to 80 days at 4 and 25 degrees C in both carriers (sludge and peat). However, for the secondary sludge-grown cells stored in peat-based carrier at 4 degrees C, the viable cells decreased under 10(8) cells/g at the 81st day of storage but remained acceptable compared to the standard (10(7) cells/g of carrier).     

Effect of Storage Temperature and Duration on Survival and Infectivity of Steinernema innovationi (Rhabditida: Steinernematidae)

Entomopathogenic nematode species differ in their optimum storage temperature; therefore, we conducted a study on the survival and infectivity of the recently described Steinernema innovationi from South Africa at five storage temperatures (5°C, 10°C, 15°C, 20°C, and 25°C) over 84 d using 20,000 infective juveniles (IJ) in 25 ml aqueous suspension containing 0.1% formalin. Our results showed that survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 d. Infectivity of IJ against Galleria mellonella larvae was >90% for all temperatures except for 5°C at which survival decreased to 10% after 84 d. In addition, we stored 2.5 million IJ on a sponge formulation in 15 ml of 0.1% formalin solution for 84 d at the optimum 15°C followed by 2 wk storage at 25°C. Storage of the IJ on a sponge formulation for 14 d at 25°C post 15°C storage for 84 d did not have a detrimental effect on IJ survival (87%) or infectivity to G. mellonella (95%).     

Development of formulations of biological agents for management of root rot of lettuce and cucumber

The effect of various carrier formulations of Bacillus subtilis and Pseudomonas putida were tested on germination, growth, and yield of lettuce and cucumber crops in the presence of Pythium aphanidermatum and Fusarium oxysporum f.sp. cucurbitacearum, respectively. Survival of B. subtilis and P. putida in various carriers under refrigeration (about 0 degree C) and at room temperature (about 22 degrees C) was also studied. In all carrier formulations, B. subtilis strain BACT-0 survived up to 45 days. After 45 days of storage at room temperature (about 22 degrees C), populations B. subtilis strain BACT-0 were significantly higher in vermiculite, kaolin, and bacterial broth carriers compared with other carriers. Populations of P. putida were significantly higher in vermiculite, peat moss, wheat bran, and bacterial broth than in other carriers when stored either under refrigeration (about 0 degree C) or at room temperature (about 22 degrees C) for 15 or 45 days. Germination of lettuce seed was not affected in vermiculite, talc, kaolin, and peat moss carriers, but germination was significantly reduced in alginate and bacterial broth carriers of B. subtilis compared to the non-treated control. Germination of cucumber seed was not affected by any of the carriers. Significantly higher fresh lettuce and root weights were observed in vermiculite and kaolin carriers of B. subtilis compared with P. aphanidermatum-inoculated control plants. Lettuce treated with vermiculite, and kaolin carriers of B. subtilis, or non-inoculated control lettuce plants had significantly lower root rot ratings than talc, peat moss, bacterial broth, and P. aphanidermatum-inoculated control plants. Growth and yield of cucumber plants were significantly higher in vermiculite-based carrier of P. putida than the other carriers and Fusarium oxysporum f.sp. cucurbitacearum-inoculated plants.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Roots of Pisum sativum L. exhibit hydrotropism in response to a water potential gradient in vermiculite

In the present study, root hydrotropism in an agravitropic mutant of Pisum sativum L. grown in vermiculite with a steep water potential gradient was examined. When wet and dry vermiculite were placed side by side, water diffused from the wet (-0.04 MPa) to the dry (-1.2 MPa) and a steep water potential gradient became apparent in the dry vermiculite close to the boundary between the two. The extent and location of the gradient remained stable between the fourth and sixth day after filling a box with vermiculite, and the steepest gradient (approx. 0.02 MPa mm-1) was found in the initially dry vermiculite between 60 and 80 mm from the boundary. When seedlings with 25-35 mm long roots were planted in the initially dry vermiculite near where the gradient had been established, each of the main roots elongated toward the wet vermiculite, i.e. toward the high water potential. Control roots elongated without curvature in both the wet and the dry vermiculite, in which no water potential gradient was detectable. These results show that pea roots respond to the water potential gradient around them and elongate towards the higher water potential. Therefore, positive hydrotropism occurs in vermiculite just as it does in air. Hydrotropism in soil may be significant when a steep water potential gradient is apparent, such as when drip irrigation is applied.     

Polysaccharide production benefits dry storage survival of the biocontrol agent Pseudomonas fluorescens S11:P:12 effective against several maladies of stored potatoes

Pseudomonas fluorescens S11:P:12 (NRRL B-21133) is a biological control agent able to suppress several potato diseases and sprouting. Notably, it produces a polysaccharide during liquid cultivation, and the objective of this work was to determine the role of this material in the bio-control process. First, the polysaccharide was isolated, purified and identified as marginalan, which accumulated to ~3.3 g/L in cultures. The bioactivity of isolated marginalan applied alone or in combination with washed cells of strain S11:P:12 was tested in potato bioassays of dry rot and pink rot suppressiveness and sprout inhibition. Since the formulation and storage of a dried biocontrol product is preferred for commercial use, the impact of marginalan on cell survival during drying and storage was also studied. Washed bacteria formulated with 0–6.6 g/L polysaccharide were either applied to Hyflo granules, then slowly dried for 24 h with airflow at 50–60% relative humidity, or in 1-µL droplets placed in replicate wells of a micro-plate, then quickly dried for 1 h in a biohazard hood. Both Hyflo and micro-plate dry storage results indicated that marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5–5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected P. fluorescens strains, its benefits to drying and storage survival were again evident (especially after 4°C instead of 25°C storage), but its effects were more subtle than for strain S11:P:12, and dry rot suppression was not impacted.     

Biological control of crown gall, Agrobacterium tumefaciens

Biological control of crown gall caused by Agrobacteriurn turnefaciens (Smith & Townsend) Conn, pioneered by Dr A. Kerr in South Australia, is effected through the establishment of a high population of the related non-pathogen A. radiobacter (Beijerinck & van Delden) Conn, strain 84 in the rhizosphere of susceptible plants. Strain 84 produces a bacteriocin to which many strains of the pathogen in Australasia, North America and Britain are sensitive. The disease is present in Britain on a variety of hosts including cherry. At East Malling cherry leaf scars, invaluable as an avenue of infection for bacterial canker infectivity titrations, have been used successfully in crown gall studies. Live cells of strain 84, but neither an avirulent strain of the pathogen nor a soil bacterium highly antagonistic to A. tumefaciens in vitro, inhibited gall formation in cherry leaf scars. Heat-killed cells had no effect. In a field experiment at East Malling hardwood cuttings of the new rootstock Colt have been dipped in strain 84 and total inhibition of crown gall is expected to ensue. The results of other experiments where the disease is already established on cherry-rootstock layer-beds and in blackberry plantations are less predictable. In time we hope to solve this problem. Only time will show whether this method of biological control is long lasting or will eventually break down.     

Biological control of crown gall in cherry rootstock propagation

English isolates of Agrobacterium tumefaciens from cherry were sensitive to the bacteriocin produced by A. radiobacter strain 84 in vitro , and simultaneous inoculation of the two organisms into tomato stems or cherry leaf scars completely inhibited the gall formation that occurred in the absence of strain 84. However, attempts to achieve biological control of crown gall of cherry in the field were successful only when the antagonist was applied as a preplanting treatment to cuttings. Disease on infected cherry layers was not reduced even after three sprays of the antagonist. A. radiobacter strain 84 was also ineffective as a preplanting dip, or both preplanting and post-harvest dips for symptomless, but latently infected, rootstocks harvested from an infected layer bed.     

Biological Control of Crown Gall: Seed Inoculation

S ummary : Peach seeds were inoculated with the nonpathogenic isolate 84 of Agrobacterium radiobacter var. radiobacter biotype 2 before sowing in natural soil heavily inoculated with the tumour inducing biotype 2 isolate 27 ( A. radiobacter var. tumefaciens ). Nonpathogens (presumably isolate 84) became predominant in the biotype 2 population on roots, on underground stems and in the soil round plant crowns. Significant ( P < 0·001) biological control of crown gall was achieved. Total gall incidence on plants grown from inoculated seed was 31% and from uninoculated seed 79%; the corresponding gall incidence on plant crowns was 12% and 76%. Dusting seed with Thiram (3·1 g/kg seed) did not significantly reduce disease incidence. Infection appeared to occur through undamaged lenticels indicating that wounding is not a necessary prerequisite for crown gall induction in peach.     

Plasmids in avirulent strains of Agrobacterium.

Twelve strains of Agrobacterium radiobacter isolated from naturally occurring crown galls or soil were found to be avirulent on sunflower, tomato, Kalanchoe, and carrot. Eleven strains contained plasmids of molecular weights 77 X 10(6) to 182 X 10(6) as determined by electron microscopy. One strain contained only a smaller plasmid (50 X 10(6) daltons). Several strains had both large and small (ca. 11 X 10(6) daltons) plasmids; one strain contained two large plasmids (112 X 10(6) and 136 X 10(6) daltons). Hybridization reactions of virulence plasmids from Agrobacterium tumefaciens strains C58 and A6 with plasmids from each of the A. radiobacter strains revealed that some A. radiobacter plasmids had less than 10% homology to either the C58 or A6 plasmids. Plasmids from some strains had approximately 50% homology with the C58 plasmid, but only one A. radiobacter plasmid contained more than 10% homology to the A6 plasmid. The presence of large plasmids in A. radiobacter strains did not correlate with sensitivity to agrocin 84; however, the utilization of the amino acid derivatives octopine and nopaline was generally correlated to partial base sequence homology to the C58 plasmid. We conclude that all large plasmids found in Agrobacterium strains are not virulence associated, although they may share base sequence homology with a virulence-associated plasmid. Further, plasmids from tumorigenic strains may be more closely related by base sequence homology to plasmids from nonpathogenic strains than to plasmids from other pathogenic strains.     

Engineering factors in single-cell protein production. II. Spray drying and cell viability

One important economical method for producing singlecell protein is to spray dry the cultured cells. This study presents some preliminary data on the effects of spray drying on cell viability. Under conditions similar to those for the production of spray-dried milk, 4–5 log cycles destruction occurred. The results indicate that, the activation energy for thermal destruction of yeast was reduced from the normal heat treatment value of 84 kcal/°K mole to about 38 kcal/°K mole.     

Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices

Lyophilization and vacuum- or spray-drying are some of the most useful techniques for preserving foods, agricultural products, and pharmaceuticals. Biological materials, however, can be irreversibly damaged during these treatments. Therefore, it is essential to design protective agents to preserve protein activity and cell viability. In this paper we examine the use of alpha, alpha-trehalose-borate systems as protectants for Lactobacillus acidophilus during freeze- and vacuum-drying. Trehalose was found to be an effective protectant for freeze-dried and vacuum-dried samples, and it is equivalent to a protective formulation which is in current industrial use. It is known from our previous work on enzymes that the presence of borate can dramatically enhance the protective ability of trehalose. In this work, the addition of trehalose-borate to bacterial concentrate greatly improves the recovery of viable cells after storage. This improvement was seen in freeze-dried samples stored at 37 degrees C as well as for vacuum-dried samples held at room temperature. A tailored buffering strategy was tested to counteract the high pH resulting from the addition of borate to the mixture. Use of citric or lactic acids in combination with ammonium hydroxide gave a protectant solution with high pH (resulting in effective crosslinking between trehalose and borate) but a dry product with reduced pH upon rehydration (conducive to cell survival). These results raise exciting possibilities for protection of more labile prokaryotic species as well as simple eukaryotes.