Biological Control of Crown Gall: Seed and Root Inoculation

S ummary . Experiments on the control of crown gall by inoculating susceptible plants with a non-pathogenic strain of Agrobacterium radiobacter have continued. In all experiments, highly significant disease control was achieved. In one experiment, 42% of untreated plants growing in soil heavily infested with A. radiobacter var. tumefaciens died; inoculation of seed with the non-pathogenic strain reduced this to nil. Combined seed and root inoculation was more efficient than seed inoculation alone. In naturally infested soil, combined seed and root inoculation at transplanting gave 99% control of gall formation (as dry weight). A significant increase in plant growth resulted from combined seed and root inoculation. At transplanting, roots should probably be inoculated within 2 h of lifting. This method of biological control is now widely practised by commercial growers in South Australia.     

Biological Control of Crown Gall: Field Measurements and Glasshouse Experiments

S ummary : An ecological study showed that the pathogeniec form of Agrobacterium radiobacter (A.r. var. tumefaciens ), biotype 2 was present in soil near galled almond seedlings, where it formed a significant proportion of the total biotype 2 population; the pathogen was not detected near healthy plants. One nonpathogenic biotype 2 isolate was able, when present in numbers approximately equal to the pathogen, to prevent crown gall induction in tomato and peach seedlings. This isolate gave complete biological control of crown gall in a glasshouse test. Six other nonpathogenic isolates did not significantly inhibit gall induction by equal numbers of the pathogen. In the field, gall induction probably depends on the size of the population of the pathogen (both absolutely and relative to that of the nonpathogen) at a wound site and on the inhibitory efficiency of the nonpathogen.     

Seed Transmission of Pea Seed-borne Mosaic Virus in Peas: Early and Late Expression of the Virus in the Progeny

Abstract Field trials at Summerland and Creston in 1984 and 1985 on loam soils naturally intested with Agrobadonum nonfactures showed that A radiobacter Strain 84 K-S4, did not control crown gall on Antonovka apple seedlingy when applied as a root-dip. K-84 controiled crown gall at Sidney on sandy loam soil m 1984 and 1985, but tailed in 1986. At Creston, higher than standard concentrations of K-84 did not control crown gall of seedlings, whether the seedlings were grown in unsterililzed soil 1986) or in sterile soil, 1987 before transplanting them into A nonfactures naturally intested soil. It a field trial in 1986 at Summerland, root-dip treatment with K-84 alone or in combination with broadeast treatments of melam-sodium of formalin tailed to control crown gall on Antonovka seedlings. However, crown gall intection was significantly reduced with metam-sodium or K-84 treatment alone when seedlings were raised in sterile soil before transplanting in naturally instead safe at Summerland in 1987.     

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.     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Population Heterogeneity of Agrobacterium tumefaciens in Galls of Populus L. from a Single Nursery

This study focused on the natural crown gall infections occurring in a Leuce poplar nursery. Soil effects on crown gall frequency were detected, indicating that contamination was due to a resident Agrobacterium tumefaciens population, which was present before seedling plantation. The crown gall frequency on poplar progenies varied from 3 to 67%, indicating the feasibility of improvement in crown gall resistance. Of 129 tumor isolates, 128 were pathogenic. These isolates were of biotype 1 or 2. Biochemical, serological, and antibiotic resistance typing results concurred, indicating the presence of four biotype 1 and two biotype 2 resident subpopulations. No significant change was noticed in the relative proportions of subpopulations from one year to another. Pathogenic subpopulations both in vitro and in planta were susceptible to Kerr K84 (P. B. New and A. Kerr, J. Appl. Bacteriol. 90:172-179, 1972). In addition, no serological cross-reactions were found to occur between K84 and the pathogenic subpopulations.     

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.     

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.     

日本樱花根癌病病原菌的鉴定及其防治

从浙江省的慈溪、奉化、嵊州等地的日本樱花苗圃内,采集到具有典型症状的日本樱花根癌病植株。经分离纯化及在指示植物番茄、向日葵幼苗的致病性测定,共分离到致病性病原菌株１１株,经形态学、生理生化学特征鉴定及菌体可溶性蛋白ＳＤＳ－聚丙烯酰胺凝胶电泳,确定引起日本樱花根癌病的病原细菌为根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａｃｉｅｎｓ）生物型１,经平皿拮抗和盆栽试验表明,生防菌Ｋ８４能够明显抑制致病菌株的致癌能力。     

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 in Grapevine and Raspberry by Two Pseudomonas spp. with a Wide Spectrum of Antagonistic Activity

Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens , when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall caused by A. tumefaciens on grapevine and raspberry. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. The disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased by 2-3.5-fold, while the DSI was reduced by 1.5-3-fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pretreatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24 or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.     

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.     

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.     

Rhizobium nepotum sp. nov. isolated from tumors on different plant species

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.     

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 × 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.     

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.     

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^‐.     

Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens.

Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group. All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present. Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA. Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline. All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics. Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains. These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested. They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal. Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA.     

Biological Control of Meloidogyne arenaria by Pasteuria penetrans

An obligate parasitic bacterium of nematode, Pasteuria penetrans 98–35 (PP), isolated from oriental melon greenhouse soil in Korea, was evaluated against root-knot nematode, Meloidogyne arenaria (MA), in tomato (Lycopersicon esculentum Mill. var. Youngkwang) and oriental melon (Cucumis melo L. var. Eunchun). Pot experiments were conducted by planting the tomato seedlings in a medium inoculated with 5,000 MA juveniles/pot (J2), J2+100,000 PP endospores/1 g medium, and J2+200,000 PP endospores/1 g medium. After 10 weeks from planting, root gall percentage in J2+100,000 and J2+200,000 PP endospores/1 g medium were significantly lower with 37.5% and 6.7%, respectively, compared with the J2 of 85%. In the second planting of tomatoes in the same pots, root gall numbers were significantly lower in PP treated pots representing 68.8 and 31.4/root in J2+100,000 and J2+200,000 PP endospores/1 g medium, respectively, compared with the J2 of 460.6/plant. In oriental melon experiment, numbers of root galls after 10 weeks from planting were significantly lower in J2+100,000 endospores/1 g medium with 32.5 compared with 64.1 and 87.5 in J2+100,000 endospores/1 g medium and the J2, respectively. However, there were no significant differences in plant growth characteristics among the treatments in the both crops.     

Dissimilatory ammonia production vs. denitrification in vitro and in inoculated agricultural soil samples

A dissimilatory ammonia-producing isolate identified as Enterobacter amnigenus and a denitrifier identified as Agrobacterium radiobacter isolated from the same soil were studied. The products of nitrate reduction in a minimal medium, enriched with glucose and containing nitrate N as the sole nitrogen source, were quantified when each of these isolates was cultured anaerobically, alone or mixed together in the presence or absence of C(2)H(2). When they were cultured together, ammonia was the principal product of nitrate reduction. The distribution between denitrification and dissimilatory ammonia production (DAP) for nonsterilised soil samples inoculated with E. amnigenus or A. radiobacter, or a mixture of these two isolates, was also investigated. Production of NH(4)(+) was increased under these conditions (strict anaerobiosis and much available fermentable carbon), but the inoculation of soil samples with 1.2?×?10(7) cells of E. amnigenus·g dried soil(-1) was not sufficient to shift nitrate reduction from nitrous oxide (denitrification) to ammonia production, suggesting that inoculation with a greater number of DAP bacteria than introduced would probably be required to enable ammonia production to exceed nitrous oxide release. Key words: dissimilatory ammonia production, denitrification, Enterobacter amnigenus, Agrobacterium radiobacter.