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.     

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.     

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。     

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.     

樱桃砧木实生苗对根癌菌侵染的抗性及生理响应

以贵州樱桃的6个砧木实生苗为材料,采用人工接种根癌菌的方法,探讨它们对根癌菌侵染的抗性及生理响应特征,评价其抗病性能力。结果显示:(1)接种根癌菌120d后,青岩樱桃实生苗发病率较低(6.67%),冠瘿瘤直径较小(1.76mm),感病系数也较低(6.67),属高抗砧木;沿河樱桃实生苗发病率高达50.0%,冠瘿瘤直径达6.43mm,感病系数达33.76,属高感砧木。(2)感染根癌菌后,各樱桃砧木幼苗的生理特性受到一定影响,其叶片游离脯氨酸含量增加,根系过氧化物酶(POD)和超氧化物歧化酶(SOD)活性增强,而过氧化氢酶(CAT)活性降低,其中,青岩樱桃的游离脯氨酸含量升高幅度最大,沿河樱桃的保护酶活性变化幅度明显较大。(3)樱桃砧木幼苗的发病率与感病系数呈显著正相关(0.98*);发病率与SOD活性、感病系数与POD活性和SOD活性均呈显著正相关,相关系数分别为0.86、0.82、0.83;其余指标间相关性不显著。研究表明,6个樱桃砧木实生苗对根癌菌的抗性有明显差异,并以青岩樱桃最强,且其抗性强弱与其感病后叶片脯氨酸含量和根系的POD、SOD活性增加幅度有密切关系。     

樱桃根癌土壤杆菌及其对土壤杆菌素84敏感性的研究

从山东、河北、辽宁等地樱桃园的樱桃冠瘿瘤和土壤样品中分离到46株根瘤土壤杆菌。经鉴定有4株是Agrobacteriumtumefaciens（原生物型1）,其余42株是A.rhizogenes（原生物型2）。这些菌株所诱导的冠瘿瘤中均合成胭脂碱（nopaline）,属胭脂碱型Ti质粒的根癌土壤杆菌,并对放射土壤杆菌K84菌株所产生的土壤杆菌素84敏感。由于K84菌株对含胭脂碱Ti质粒的根癌土壤杆菌有很好的抑制效果,因此,用K84菌株防治樱桃根癌病是有应用前景的．     

Construction of a Range of Derivatives of the Biological Control Strain Agrobacterium rhizogenes K84: a Study of Factors Involved in Biological Control of Crown Gall Disease

The biological control strain Agrobacterium rhizogenes K84 is an effective agent in the control of Agrobacterium pathogens, the causative agents of crown gall disease. A number of factors are thought to play a role in the control process, including production of the specific agrocins 84 and 434, which differ in the spectra of pathogenic strains that they inhibit in vitro. A range of derivatives of strain K84 has been developed with every combination of the three resident plasmids, pAgK84, pAgK434, and pAtK84b, including a plasmid-free strain. These derivatives produced either both, one, or neither of the characterized agrocins 84 and 434 and were isolated by plasmid curing, conjugation, and Tn5 transposon mutagenesis. The ability of the derivative strains to inhibit gall formation on almond roots was compared to that of the wild-type K84 parent. Treatment with the plasmid-free derivative did not result in a significant level of control of an A. rhizogenes pathogen based on numbers or dry weight of galls formed on injured almond roots. The presence of plasmid pAgK84, pAgK434, or pAtK84b significantly enhanced the biological control efficacy of K84 derivatives, and the highest level of control was observed with strains harboring two or more plasmids. The results observed with strains deficient in agrocin 434 production suggest that this product may play an important role in the biological control of A. rhizogenes pathogens. The involvement of plasmid pAgK84b in biological control has not previously been reported. This study supports the conclusion that multiple factors are involved in the success of strain K84 as a biological control agent.     

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.     

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.     

Evidence for the Presence of Bacteria-specific Proteins in Sterile Crown Gall Tumor Tissue

Cross-reacting antigens were found in bacteria-free crown gall tumor tissue tested with serum prepared against Agrobacterium tumefaciens (Smith and Towns.) Conn., but no such antigens were detected in callus tissue. Soluble proteins from tumor tissue, callus tissue, and the crown gall bacteria were fractionated on a DEAE-Sephadex (A-50) column. The diethylaminoethyl-Sephadex elution profile for tumor tissue showed three protein fractions that were not detected in the callus tissue. Two of these protein fractions were shown to be exclusively bacteria specific. Besides these qualitative differences between the two tissues, significant quantitative differences in the amount of protein fractions were also observed. The diethylaminoethyl-Sephadex column fractions from tumorigenic strain of A. tumefaciens corresponding in position to the three additional peaks in the tumor tissue also showed cross-reacting antigens when tested with serum prepared against sterile tumor tissue. It is suggested that tumor formation by A. tumefaciens involves integration of the bacterial genome into the host-cell genome.     

Evaluation of Biological and Chemical Treatments for Control of Crown Gall on Young Apple Trees in the Kootenay Valley of British Columbia

Field trials at Creston in 1988, 1989, and 1990 on silty loam soil naturally infested with Agrobacterium tumefaciens showed that Agrobacterium radiobacter strain K84 did not control crown gall on young Antonovka apple trees when apphed as a root-dip treatment. Copper oxychloride applied as a root-dip treatment at 2.5 and 5.0 g ai/1 and sewage sludge applied at 260 g per tree as broadcast were effective in reducing crown gall infection but these treatments were toxic to young apple trees in 1989. Lower rates of copper oxychloride, 0.3, 0.6 and 0.9 g ai/1, and sewage sludge at 130 g per tree, did not control crown gall in the 1990 field trial. The biological treatment with the strain AB8 of Bacillus subtilis applied as a root-dip effectively controlled crown gall and was not phytotoxic to young Antonovka apple trees. These results suggest that strain AB8 of B. subtilis has the potential to control crown gall on young apple trees under field conditions.     

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.     

Systemic Migration of Pseudomonas syringae pv. avellanae in Twigs and Young Trees of Hazelnut and Symptom Development

At the beginning of October 1992 and 1993, healthy adult and young trees of hazelnut, cultivars Tonda Gentile Romana and Nocchione, were inoculated with a Pseudomonas syringae pv. avellanae strain through leaf scars located midway from the tip of the twig. In adult trees, the systemic migration and the population level of the pathogen were monitored during autumn and winter by means of weekly to monthly isolation and countings. In young plants, the possible displacement of the bacterium was checked in May by sampling different sites of the plant. At each sampling, the presence of internal and external symptoms was carefully assessed. During the 2-year study, the results on migration and symptom development were consistent between the cultivars. Until the end of February. P.s. pv. avellanae colonized for approximately 45 mm, only the trait of the twig below the site of inoculation. At that time the population level ranged, in the farthest point reached by the pathogen, from 4.1 to 5.6 = 10⁴ cfu/3 mm of twigportion. During March the pathogen was recovered from the tip of the twig. The first sign of disease was noticed in November as brown discolouration of the epidermis near the site of inoculation as well as internal necrosis of the vascular tissue. In young trees. P.s. pv. avellanae from the twig reached the branches, collar and roots from where it also colonized the branch which did not carry an inoculated leaf scar. Extensive twig dieback was noticed during April, whereas in June, most of the adult trees inoculated in autumn with approximately 25000 cells of the pathogen, randomly distributed in 25 sites of the crown, wilted completely. The role of leaf scars as a favourite site of penetration exploited by the pathogen as well as phytosanitary measures are discussed.     

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes

Background

Agrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of α-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here.

Results

TFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio.

Conclusions

TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.     

Bacterial canker of stone-fruits: Infection experiments with Pseudomonas mors-prunorum and P. syringae

The effect was compared of inoculating Pseudomonas mors-prunorum and P. syringae at high inoculum concentration through wounds in plum stems and cherry branches, and through cherry-leaf scars. In the wound inoculations a North American cherry strain of P. syringae was considerably more virulent than any of three indigenous cherry strains of P. mors-prunorum, but two pear strains of P. syringae were less virulent. P. mors-prunorum showed a much greater capacity to invade through leaf scars, particularly towards the end of the leaf-fall period, when all three P. syringae strains were largely ineffective. Evidence is discussed which suggests that the greater infectivity of the P. mors-prunorum strains through leaf scars was related to their capacity to colonize the host tissues from small inocula. The P. syringae strains died out in cherry cankers earlier than P. mors-prunorum strains and they were less stable during host passage. The results of the experiments indicate that virulence in these organisms is a complex phenomenon and determined by several factors.     

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.     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.     

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.     

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.     

New Antagonistic Strains of Non‐Pathogenic Agrobacterium vitis to Control Grapevine Crown Gall

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded non‐pathogenic strains of Agrobacterium. On the basis of classic diagnostic tests, a sequence analysis and a previously reported multiplex PCR method, the non‐pathogenic strains ARK‐1, ARK‐2 and ARK‐3 were identified as Agrobacterium vitis. Stems of grapevine seedlings were inoculated with both a cell suspension of seven mixed strains of A. vitis (Ti) as a pathogen and one of a new strain or A. vitis strain VAR03‐1, one of the biological control agents against crown gall previously reported, as competitors to assay the suppression of tumour formation caused by the pathogen. In a test with a 1:1 cell ratio of pathogen/nonpathogen, strains ARK‐1, ARK‐2 and ARK‐3 reduced the tumour incidence.. In particular, strain ARK‐1 was strongest at inhibiting tumour formation in this study. Strain ARK‐1 established populations on roots of grapevine tree rootstock and persisted on roots for a year. ARK‐1, ARK‐2 and ARK‐3 did not produce a halo of inhibition against A. vitis (Ti) strain on YMA medium. Moreover, strain ARK‐1 did not reduce tumour incidence on the stems of grapevine when ARK‐1 was dead or only culture filtrate was used. This result indicates the possibility that these new strains inhibit grapevine crown gall in planta by a different mechanism other than VAR03‐1. In particular, one of the new strains, named ARK‐1, was most effective in inhibiting tumour formation on grapevine and appears to be a promising new agent to control grapevine crown gall.