Effect of antibiosis on antagonist dose-plant disease response relationships for the biological control of crown gall of tomato and cherry

ABSTRACT The crown gall pathosystem was used to evaluate a model that describes the dose-response relationship between biological control agents and plant pathogens. The model predicts that this relationship can become asymptotic, such that increased antagonist doses cannot compensate for deficiencies in disease suppression. Wounded roots of tomato (Lycopersicon esculentum) and cherry (Prunus mahaleb) plants were dipped into different concentrations of the biological control organism Agrobacterium radiobacter strain K84 prior to inoculation with the pathogen A. tumefaciens. Pathogen strains sensitive or resistant to the antibiotic agrocin 84 were used, and for tomato experiments, a derivative of A. radiobacter strain K84 that does not produce agrocin 84 also was included as an experimental treatment. As predicted by the dose-response model, the amount of disease suppression per unit of antagonist decreased with increasing antagonist dose and became asymptotic at high antagonist densities. Control of crown gall of tomato was nearly complete with the combination of A. radiobacter K84 and an agrocin 84-sensitive strain of A. tumefaciens. Pathogen resistance to agrocin 84 or lack of agrocin 84 production by A. radiobacter resulted in antagonist dose-crown gall incidence relationships that were apparently asymptotic at levels of control significantly less than 100%. For field-grown cherry, similar dose-response relationships were observed with higher asymptotic levels of disease suppression obtained when trees were inoculated with an agrocin 84-sensitive A. tumefaciens strain compared with an agrocin 84-resistant pathogen strain. The differences among bacterial strain combinations in the magnitude of the asymptote defined by the dose-response relationships suggest that A. radiobacter impacts a smaller proportion of the pathogen population when the activity of agrocin 84 is muted.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

Results of seven years of biological control of Agrobacterium tumefaciens in Spain1

Biological control of Agrobacterium tumefaciens using the K84 strain of A. radiobacter has been practised in Spain since 1979. Strain K84 is produced in Sevilla by the Agricultural Research Service and is mixed with peat. This formulation is authorized for use on stone fruits and roses, since results of various trials on rootstocks of these crops have shown that K84 is effective in controlling the disease in naturally infected nursery soil, as well as in experimental tests carried out on artificially inoculated soil. Results of biological control trials have shown that K84 has some effect on A. tumefaciens strains resistant to agrocin 84. Factors other than susceptibility to agrocin 84 influence the efficacy of biological control. This was demonstrated by using a K84 agrocin non-producer strain which proved effective against both resistant and susceptible strains. Biological control on apple or pear is not yet authorized in Spain due to a paucity of data on field trials. However, initial pot tests showed good K84 activity on these hosts, against Spanish strains of A. tumefaciens. On grapevine, most of the A. tumefaciens strains that were isolated belong to biotype 3 and are resistant to agrocin 84. Because the disease is systemic on this species the use of K84 is not authorized.     

Monitoring of spirodiclofen susceptibility in field populations of European red mites, Panonychus ulmi (Koch) (Acari: Tetranychidae), and the cross-resistance pattern of a laboratory-selected strain

BACKGROUND: Phytophagous mites such as the European red mite, Panonychus ulmi (Koch), are serious pests in European fruit tree orchards, and a number of acaricides are frequently used to control them. Spirodiclofen (Envidor^®) has been a commonly used acaricide for several years. In the present study, European field populations collected in 2009 and 2010 were checked for their susceptibility to spirodiclofen by using discriminating dose and full dose response bioassays. RESULTS: In 2009 and 2010, a total of 63 field populations (including winter eggs) of European red mites were collected in different European countries, and in several populations from south‐western Germany a shifting in susceptibility against spirodiclofen was observed. Full dose response bioassays on different developmental stages of field‐collected strains suggested an age‐dependent expression of resistance because eggs remain fully susceptible to spirodiclofen. Artificial selection with spirodiclofen of one of the field strains resulted in resistance ratios of > 7000. Synergism studies suggest a possible role of cytochrome‐P450‐dependent monooxygenases in spirodiclofen detoxification. Most of the other acaricides from different chemical classes displayed no or low cross‐resistance in a spirodiclofen‐selected strain. CONCLUSION: In order to preserve spirodiclofen as an important tool in spider mite resistance management, the efficacy situation should be continuously monitored, and it is suggested that spirodiclofen be alternated with acaricides coming from different mode‐of‐action classes. An observed age‐specific expression of resistance revealed full susceptibility of eggs, so targeting spirodiclofen particularly against eggs is likely to reduce the selection pressures imposed on other life stages. Copyright © 2011 Society of Chemical Industry     

Application of alginate gel for protection of wounds against crown gall (Agrobacterium tumefaciens)

In glasshouse and laboratory experiments, calcium alginate gel, produced in situ on wounds by successive dipping in solutions of sodium alginate and CaCl₂ gave significant protection against infection by pathogenic isolates of Agrobacterium tumefaciens on carrot discs, tomato cuttings and chrysanthemum cuttings. Gel protection was sometimes equal to that given by strain K84 of A. radiobacter and also operated against a pathogenic isolate insensitive to control by strain K84. Sodium alginate or CaCl₂ alone gave little or no protection, suggesting that calcium alginate acted by physically excluding the pathogen from wound surfaces. The use of the method described is discussed for delivery of biological or chemical control agents to plant surfaces whilst also providing protection in its own right.     

Biological control of weeds in European crops: recent achievements and future work

Approaches to the biological control of weeds in arable crops and integration of biological weed control with other methods of weed management are broadly discussed. Various types of integrative approaches to biological control of weeds in crops have been studied within the framework of a concerted European Research Programme (COST‐816). During the period 1994–99, some 25 institutions from 16 countries have concentrated on five target weed complexes. Some major scientific achievements of COST‐816 are: (i) combination of the pathogen Ascochyta caulina with an isolated phytotoxin produced by this fungus to control Chenopodium album in maize and sugar beet; (ii) the elaboration and preliminary field application of a system management approach using the weed:pathogen system Senecio vulgaris:Puccinia lagenophorae to reduce the competitiveness of the weed by inducing and stimulating a disease epidemic; (iii) combination of underseeded green cover with the application of spores of Stagonospora convolvuli to control Convolvulus species in maize; (iv) assessment of the response of different provenances of Amaranthus spp. to infection by Alternaria alternata and Trematophoma lignicola, the development of formulation and delivery techniques and a field survey of native insect species to control Amaranthus spp. in sugar beet and maize; (v) isolation of strains of different Fusarium spp. that infect all the economically important Orobanche spp. and development of novel, storable formulations using mycelia from liquid culture. Although no practical control has yet been reached for any of the five target weeds, potential solutions have been clearly identified. Two major routes may be followed in future work. The first is a technological approach focusing on a single, highly destructive disease cycle of the control agent and optimizing the efficacy and specificity of the agent. The second is an ecological approach based on a better understanding of the interactions among the crop, the weed, the natural antagonist and the environment, which must be managed in order to maximize the spread and impact of an indigenous antagonist on the weed.     

Evidence of pAgK84 transfer from Agrobacterium rhizogenes K84 to natural pathogenic Agrobacterium spp. in an Italian peach nursery

Nine Italian peach nurseries, which use Agrobacterium rhizogenes strain K84 to protect plants from crown gall, were monitored for three years with the aim of determining whether transconjugant populations may arise following plasmid exchanges between K84 and autochtonous soil agrobacteria. Six hundred and seventy-eight Agrobacterium isolates were obtained from 120 tumours developed on apricot and peach rootstocks that had been treated in pre-planting with the antagonist. Agrobacteria were characterized for pathogenicity, biovar, opine catabolism and agrocin 84 sensitivity. Colony hybridization was used for screening the isolates harbouring plasmids pTi and/or pAgK84. Analysis of plasmid content and Southern blotting were performed on putative transconjugant agrobacteria found in tumours collected from one nursery where a biological control breakdown was observed. The RFLP analysis of 16S + IGS regions showed that pAgK84 was transferred from the antagonist to virulent and avirulent soil agrobacteria belonging to different ribotypes. Pathogenic transconjugants, inoculated on GF677 rootstocks, were not controlled in vivo by K84 and stably maintained pTi and pAgK84 in the bacterial cells for at least one year. At the end of a biocontrol trial, new transconjugant tumorigenic agrobacteria originated by the transfer of pAgK84 to the pathogen. Virulent and avirulent transconjugants may represent a real threat for biological control by K84 strain since all of them produced agrocin and were insensitive to it. Survival in soil of these populations could make the future application of K84 ineffective.     

Use of the Genetically Engineered Agrobacterium Strain K1026 for Biological Control of Crown Gall

Strain K84 of Agrobacterium (formerly called A. radiobacter) has been a successful biocontrol agent of crown gall disease for almost 30 years all over the world. In spite of its demonstrated efficiency, the most important risk of failure when using strain K84 is the possibility of transfer of plasmid pAgK84 to pathogenic Agrobacterium strains. pAgK84 codifies production of and immunity to agrocin 84, the main factor involved in crown gall biocontrol by strain K84. Then, a second generation of strain K84 was obtained and the genetically engineered strain was called K1026. It contains a deletion in the transfer region of pAgK84. To date, a considerable number of studies have been performed to compare both strains in its ability to control crown gall, plasmid transfer, antibiotic production, root colonization and survival in the rhizosphere. The aim of this review is to discuss all this comparative available information which advises that strain K1026 should be used as a biopesticide to safeguard biocontrol of crown gall wherever strain K84 is employed.     

啤酒花根癌土壤杆菌生物型的鉴定及生物防治的初步探讨

啤酒花根癌病在我国发生普遍,是影响啤酒花生产的重要病害。从啤酒花主要生产基地新疆、山东、浙江等地分离到61个菌株。通过培养性状、染色反应,以及14～15个菌株的生理生化性状和12个菌株致病性的测定,确定啤酒花根癌病病原菌为根癌土壤杆菌Agrobacteria tumefaciens(E.F.Smith &Jownsend)Conn属生物型1、病原菌菌株对K84(Agrobacterium radiobacter)菌株的细菌素敏感,各菌株间敏感性的强弱不一。盆裁番茄和啤酒花初步生防试验表明,用K84与敏感性强的菌株按1:1比例混合接种,番茄和啤酒花都不产生瘿瘤;而敏感性弱的菌株需用3:1才能防止瘿瘤的产生。     

稻瘟病菌一假定几丁质酶多克隆抗体的制备及其检测

 为检测几丁质酶的表达,制备稻瘟病菌的一假定几丁质酶多克隆抗体,探索其可能运用。将稻瘟病菌(Magnaporthe oryzae) 几丁质酶基因克隆入融合表达载体pET\|32a,并在大肠杆菌Escherichia coli BL21(DE3)中进行诱导表达。表达菌株经0.5 mmol/L IPTG诱导4～6 h后,用Ni²⁺\|NTA亲和柱纯化蛋白,得到可溶的几丁质酶\|His融合蛋白。以该融合蛋白免疫新西兰雄兔,制备多克隆抗体。ELISA分析表明该抗体效价达1∶204 800,特异性良好。用该抗体ELISA检测了稻瘟病菌4个不同田间菌株中,不同的菌株表达量不同。对稻瘟病菌侵染水稻3、5和7d后的病叶进行抗原Western验证,结果表明,稻瘟病菌不同的侵染时间其几丁质酶表达量有明显差异。 几丁质酶表达的差异是否与菌株间致病型等生物学和生理学差异有关,目前正在进一步研究。     

Effects of antagonist cell concentration and two-strain mixtures on biological control of fusarium dry rot of potatoes

ABSTRACT Eighteen bacterial strains were individually assayed against Gibberella pulicaris (5 x 10(5) conidia per ml) by coinoculating antagonist and pathogen in wounds in cv. Russet Burbank potatoes. All antagonist concentrations (10(6), 10(7), and 10(8) CFU/ml) decreased disease (38 to 76% versus control, P < 0.05). When four strains were assayed at 11 concentrations (range 10(5) to 10(8) CFU/ml) against G. pulicaris, linear regression of the log-dose, log-response data was significant for all four strains (P < 0.001 to 0.01, R(2) = 0.50 to 0.74). Challenging G. pulicaris with all possible antagonist pairings within 2 sets of 10 antagonist strains (5 x 10(5) CFU of each strain per ml) resulted in 16 of 90 pairs controlling disease better than predicted based on averaging the performance of the individual strains making up the pair (P < 0.10). Successful pairs reduced disease by ~70% versus controls, a level of control comparable to that obtained with 100 times the inoculum dose of a single antagonist strain. Neither strain genus nor soil of origin were useful in predicting successful antagonist pairs. Factors potentially influencing dose-response relationships and the effectiveness of antagonist pairs in controlling disease are discussed.     

Durability of Resistance and Cost of Virulence

A seasonal model, where a growing season is defined as the time between sowing and harvest and alternates with an inter-crop period, was derived to study the effects of the ‘cost of virulence’ and cropping ratio on durability of resistance. We assumed a single strain of virulent pathogen, a single strain of avirulent pathogen and two cultivars (one resistant and one susceptible) and studied two measures of durability of resistance (‘take-over time’ and ‘usefulness time’). Take-over time is defined as the time needed for the virulent strain of the pathogen to reach a preset threshold and predominate over the previous pathogen population. Usefulness time is the time needed before the estimated gain in green canopy area duration per plant through the use of the resistant cultivar becomes negligible. The model suggested that, although it could take several seasons before the virulent strain of the pathogen predominated over the previous pathogen population, the usefulness time of the resistant cultivar was always much shorter. Furthermore, increasing selection for the virulent strain of the pathogen (through increasing the cropping ratio of the resistant cultivar) caused the virulent strain of the pathogen to invade the system more rapidly. Cost of virulence, reflecting differences in pathogen infection rates between the four possible combinations of cultivar/pathogen strain, significantly affected durability of resistance, with the dynamics of the virulent and avirulent strains ranging from a case where the virulent strain of the pathogen died out to a case where the virulent strain of the pathogen invaded the resident pathogen population. An intermediate state, where the system reached equilibrium and the virulent strain of the pathogen neither became predominant nor died out, was defined as ‘coexistence’ of both strains of the pathogen. Occurrence of coexistence was directly related to the cost of virulence since it did not occur when virulence of the pathogen did not have a fitness cost. Two methods to include cost of virulence in the model gave similar results in relation to the two measures of durability of resistance studied.     

Improved Resistance Management for Durable Disease Control: A Case Study of Phoma Stem Canker of Oilseed Rape (Brassica napus)

Specific resistance loci in plants are generally very efficient in controlling development of pathogen populations. However, because of the strong selection pressure exerted, these resistances are often not durable. The probability of a resistance breakdown in a pathosystem depends on the evolutionary potential of the pathogen which is affected by: (i) the type of resistance (monogenic and/or polygenic), (ii) the type of reproduction of the pathogen (sexual and/or asexual), (iii) the capacity of the pathogen for dispersal, (iv) the resistance deployment strategy (pyramiding of specific resistances, mixture of cultivars, spatio-temporal alternation), (v) the size of the pathogen population, which is affected by control methods and environmental conditions. We propose the concept of Integrated Avirulence Management (IAM) to enhance the durability of specific resistances. IAM involves a strategy to limit the selection pressure exerted on pathogen populations and, at the same time, reduce the size of pathogen populations by combining cultural, physical, biological or chemical methods of control. Several breakdowns of resistance specific to Leptosphaeria maculans, the causal agent of phoma stem canker have occurred in Europe and in Australia. This review paper examines control methods to limit the size of L. maculans populations and discusses how this limitation of population size can enhance the durability of specific resistances. It proposes pathways for the development of a spatially explicit model to define IAM strategies. Simulation results are presented to demonstrate the potential uses of such a model for the oilseed rape/L. maculans pathosystem.     

K84的生物防治效果与土壤杆菌Ti质粒类型的关系

用K84对我国不同寄主来源的68个Agrobacterium tumefaciens菌株进行生物防治试验,结果表明:K84对我国桃树菌株的防治效果显著,对新疆的啤酒花菌株也有效;但对浙江萧山的部分啤酒花菌株无效;对甜菜菌株的防治效果不佳;对葡萄菌株基本上无效。对致病菌株的质粒类型的研究结果表明:桃树、啤酒花和甜菜菌株基本上都是胭脂碱(Nopaline)型质粒、葡萄菌株基本上是章鱼碱(Octopine)型质拉。试验中还发现不少胭脂碱型菌株不能被K84所防治。此外,在甜菜和毛白杨菌株中也发现了对细菌素A84(agrocin84)敏感,但不能被K84防治的菌株。     

The dose rate debate: does the risk of fungicide resistance increase or decrease with dose?

This paper reviews the evidence relating to the question: does the risk of fungicide resistance increase or decrease with dose? The development of fungicide resistance progresses through three key phases. During the ‘emergence phase’ the resistant strain has to arise through mutation and invasion. During the subsequent ‘selection phase’, the resistant strain is present in the pathogen population and the fraction of the pathogen population carrying the resistance increases due to the selection pressure caused by the fungicide. During the final phase of ‘adjustment’, the dose or choice of fungicide may need to be changed to maintain effective control over a pathogen population where resistance has developed to intermediate levels. Emergence phase: no experimental publications and only one model study report on the emergence phase, and we conclude that work in this area is needed. Selection phase: all the published experimental work, and virtually all model studies, relate to the selection phase. Seven peer reviewed and four non‐peer reviewed publications report experimental evidence. All show increased selection for fungicide resistance with increased fungicide dose, except for one peer reviewed publication that does not detect any selection irrespective of dose and one conference proceedings publication which claims evidence for increased selection at a lower dose. In the mathematical models published, no evidence has been found that a lower dose could lead to a higher risk of fungicide resistance selection. We discuss areas of the dose rate debate that need further study. These include further work on pathogen‐fungicide combinations where the pathogen develops partial resistance to the fungicide and work on the emergence phase.     

Production and preliminary characterization of monoclonal antibodies raised against <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">mangiferaeindicae</Emphasis>

Bacterial black spot disease of mango is caused by Xanthomonas campestris pv. mangiferaeindicae (Xcm), which consists of two genotypically and phenotypically distinct groups of strains. Monoclonal antibodies (MABs) were produced – 15 against CFBP 1717, a group I strain, and 9 against CFBP 2919 (yellow-pigmented), a group II strain – and were analyzed for their characteristics. On the avidin-biotin peroxidase complex enzyme-linked immunosorbent assay, the dilution limit of the MABs was between 100 and 200000 and was 10 times higher when measured on the corresponding ascitic fluid. All kinds of isotypes were represented among the MABs. All the Japanese Xcm strains, designated group I by hrp-restriction fragment length polymorphism (RFLP) analysis, reacted equally with MAB 1A7H12G3, which is the most specific for all but one worldwide group I strains, and to only one strain among group II. Also, to various extents, serological heterogeneity inside the two groups was consistently differentiated based on isozyme and RFLP analyses. MAB 1E2E1 against CFBP2919, because of its narrow specificity, and MAB 1A7H12G3 against CFBP1717, because of its broad specificity, will be useful for epidemiological studies or general control of the pathogen.     

Biological control of potato soft rot caused by Dickeya solani and the survival of bacterial antagonists under cold storage conditions

Dickeya and Pectobacterium are responsible for causing blackleg of plants and soft rot of tubers in storage and in the field, giving rise to losses in seed potato production. In an attempt to improve potato health, biocontrol activity of known and putative antagonists was screened using in vitro and in planta assays, followed by analysis of their persistence at various storage temperatures. Most antagonists had low survival on potato tuber surfaces at 4 °C. The population dynamics of the best low-temperature tolerant strain and also the most efficient antagonist, Serratia plymuthica A30, along with Dickeya solani as target pathogen, was studied with TaqMan real-time PCR throughout the storage period. Tubers of three potato cultivars were treated in the autumn with the antagonist and then inoculated with D. solani. Although the cell densities of both strains decreased during the storage period in inoculated tubers, the pathogen population was always lower in the presence of the antagonist. The treated tubers were planted in the field the following growing season to evaluate the efficiency of the bacterial antagonist for controlling disease incidence. The potato endophyte S. plymuthica A30 protected potato plants by reducing blackleg development on average by 58.5% and transmission to tuber progeny as latent infection by 47–75%. These results suggest that treatment of potato tubers with biocontrol agents after harvest can reduce the severity of soft rot disease during storage and affect the transmission of soft rot bacteria from mother tubers to progeny tubers during field cultivation.     

Aerial Contamination of Sugarcane in Guadeloupe by Two Strains of Xanthomonas albilineans

Two sugarcane plots were set up in Guadeloupe with disease-free tissue cultured plants in a banana growing location distant from sugarcane fields. Thirteen weeks after planting sugarcane in the field, a Xanthomonas albilineans strain belonging to serotype 3 (strain XaS3) was detected in water sampled at sunrise on the leaves in the first plot. This strain randomly invaded the sugarcane canopy. Seven weeks later, a new strain belonging to serotype 1 (strain XaS1) appeared on leaves and populations of strain XaS1 progressively increased on the leaf surface, whereas populations of strain XaS3 progressively decreased. Leaf scald symptoms were first noted 26 weeks after sugarcane planting. However, only strain XaS1 was isolated from leaves and a few sugarcane stalks showing symptoms. Both strains also colonized the second field plot, which was studied at the end of the experiment to avoid human interference of aerial contamination of sugarcane. After inoculation of three sugarcane cultivars by the decapitation technique, strain XaS1 was as virulent or more virulent than five other strains of X. albilineans isolated from diseased sugarcane plants in Guadeloupe. Although strain XaS3 colonized a few stalks, it failed to produce any symptoms and was the least virulent strain. Leaf surface colonization by X. albilineans was reproduced in a greenhouse trial by spraying the pathogen on sugarcane foliage. After 8 weeks, the pathogen was isolated from disinfected leaf blades. Although the leaf scald pathogen is thought to be mainly transmitted by infected cuttings, aerial transmission of X. albilineans is also known to occur. These results indicate the importance of sugarcane phyllosphere colonization by virulent strains in the epidemiological cycle of leaf scald disease in Guadeloupe.