DEPARTMENT for ENVIRONMENT, FOOD and RURAL AFFAIRS CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)
Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit DEFRA, Area 301 Cromwell House, Dean Stanley Street, London, SW1P 3JH. An electronic version should be e-mailed to [email protected]
Project title Improved control of novel Agrobacterium induced diseases in hydroponic crops through risk assessment and biological control.
DEFRA project code HH2308SPC
Contractor organisation Central Science Laboratory and location York YO41 1LZ
Total DEFRA project costs £ 150, 000
Project start date 01/01/02 Project end date 31/12/04
Executive summary (maximum 2 sides A4)
Objectives 1 & 2 To assess the potential for spread of the pRi into other micro-organisms and develop an in vitro root culture assay to screen pRi harbouring strains for pathogenicity Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harbouring a root-inducing Ri plasmid (pRi). Nine other pRi-harbouring -Proteobacteria were isolated from root mat infected crops. Three of these strains were identified as species within Ochrobactrum, five as Rhizobium, and one strain as Sinorhizobium. An in vitro pathogenicity test, inoculating cucumber cotyledons, was developed. All pRi-harbouring -Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) were lower than those observed from rhizogenic A. radiobacter (64%). However individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with the rhizogenic Sinorhizobium (75%). This is the first report of the isolation direct from environmental samples of non-Agrobacterium strains which harbour a Ri plasmid. A biocontrol agent that specifically targets A. radiobacter may fail to control root mat being induced by non-Agrobacterium strains harbouring pRi. Thus biocontrol agent(s) with a broad range of action / protection may be the best avenue of success.
Objective 3. To assess the variability of the Agrobacterium Ri-plasmid in root mat associated, and other, strains. Root-inducing Ri plasmids from seventeen -Proteobacteria strains associated with root mat of cucumbers and tomatoes were characterised by PCR-RFLP fingerprinting. Plasmids of fifteen were shown to be cucumopine Ri-plasmids. Two regions of these plasmids were selected for PCR-RFLP fingerprinting, the T- DNA, the region which is transferred to plant genomes and the vir region, genes from which mediate transfer of the T-DNA from the bacterium to the plant. PCR-RFLP analysis of four T-DNA regions and also the virD2 gene of the cucumopine Ri-plasmids indicated that these 15 plasmids were of the same type, including plasmids harboured by Agrobacterium radiobacter strains isolated in the 1970s - the first recorded outbreak of
CSG 15 (Rev. 6/02) 1 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
root mat in cucumbers, from strains isolated from both cucumbers and tomatoes, from other non- Agrobacterium sp., and from both UK and French isolates. Two other Ri-plasmids associated with a root mat outbreak in tomato were shown to belong to another, unknown, Ri-plasmid type.
Objective 4. To isolate bacteriophages, and also identify other novel classes of control agents, capable of controlling or eliminating pathogenic Agrobacterium sp. Three large scale replicated glasshouse trials were conducted at the Stockbridge Technology Centre (STC) to evaluate classes of biological control agents, and other compounds and products, plus novel growth substrates which could control or suppress the root mat pathogen.
Trial 1. Fluorescent Pseudomonas spp. have previously been shown to provide protection against a range of cucumber diseases. These bacteria, along with bacteria with biocontrol activity from other genera such as Bacillus and Stenotrophomonas, are sometimes termed as Plant Growth-Promoting Rhizobacteria (PGPR). One hundred and eighty one fluorescent Pseudomonas strains were isolated and identified by fatty acid profiling from cucumber and tomato samples. Sixteen of these strains (of 31 tested) showed the ability to prevent root mat symptoms in the in vitro pathogenicity assay. Four of these strains were evaluated in combination and as single applications, together with a commercial product Bioyield™, available in the USA and which contains two Bacillus spp. known to induce a plant growth response, in the first large scale cucumber trial. Cucumbers were grown hydroponically in rockwool.
In this trial all strains, tested individually and in combination, showed a suppression in root mat symptoms when compared to the inoculated control. At 9 and 10 weeks this difference was significant (Duncan’s test; p <0.05), indicating that increasing microbial diversity in rockwool substrates can prevent root mat infection. Disease severity was reduced by 93% (in week 10) by the best treatment, an isolate of Pseudomonas.
Trial 2. In the second STC trial the ability of a variety of other organisms, compounds and products to suppress root mat was evaluated, along with a fluorescent Pseudomonas strain shown to induce disease suppression in the first glasshouse trial. The following agents were evaluated, Agrobacterium bacteriophages, β-Aminobutyric acid (BABA) - a known inducer of plant defence mechanisms, a product (Rhizotonic) claimed to promote rhizosphere health; Pasteurised soil.
In this trial no significant suppression of root mat was observed in rockwool grown cucumbers. At the end of this trial 29% of plants had largely died from Mycospharella stem rot, compared with less than 3% in the first, which may have affected results. Root mat symptoms in plants treated with BABA, Rhizotonic, and phage were less severe than those observed in the inoculated control plants.
Trial 3. A tomato experiment was also set up to evaluate if organic growth substrates could suppress root mat symptoms when compared to plants in rockwool. The three products tested, one made of composted bark and two made of coir fibres, failed to suppress root mat symptoms when compared to plants grown in rockwool.
Objective 5 To monitor the survival of Agrobacterium and the Ri-plasmid The pathogen was found to be associated with a variety of weed species (e.g chickweed, groundsel, dandelion and soils isolated from inside and outside affected glasshouse indicating possible sources of continued infection.
Objective 6 – Technology Transfer Growers were kept informed of progress through the involvement of consultants in both the tomato and cucumber nurseries. A project application, to further the findings of the biological control work, is currently being considered by the HDC. One scientific paper has been published and one talk was presented at the 25 th Crown Gall conference held at the University of Illinois in August 2004.
CSG 15 (Rev. 6/02) 2 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Scientific report (maximum 20 sides A4) Since the early 1990s hydroponic cucumber and tomato crops in the United Kingdom and some other European countries have been affected by a disorder known as root mat. In affected crops symptoms are expressed as extensive root proliferation within rockwool cubes and slabs with, in severe cases, losses in marketable yield. An earlier outbreak of root mat in the 1970s, in soil and straw bed cucumber crops, had been associated with the presence of Agrobacterium biovar 1 strains (A. radiobacter). In the late 1990s surveys of affected crops, and subsequent host tests using Agrobacterium strains isolated during the surveys, showed that the disease in cucumbers, and a similar disorder in tomatoes, was caused by Agrobacterium radiobacter strains harbouring a root-inducing Ri plasmid (pRi) (Weller et al, 2000a; Weller et al, 2000b). Root mat symptoms are induced following transfer and expression of a segment of pRi DNA (T-DNA) into the plant cell genome, in a similar manner to that seen by plant tumour (crown gall) inducing Ti-plasmids (pTi). The T-DNA of pRi and pTi in transformed plant tissues encode genes that induce cell proliferation and also synthesis genes for unusual amino acid derivatives or sugar-phosphodiesters. These compounds are termed opines and are catabolised by genes also present on the infecting pRi or pTi. The opine associated with root mat associated pRi has been termed cucumopine.
Objective 1. To assess the potential for spread of the pRi into other micro-organisms Objective 2. To develop an in vitro root culture assay to screen pRi harbouring strains for pathogenicity
Introduction
During the surveys of the late 1990s bacterial strains had been isolated from cucumber and tomato root samples plated onto either Nutrient Dextrose (ND) medium or an Agrobacterium biovar 1 semi-selective medium, and identified by fatty acid profiling. Strains with fatty acid profiles similar to those obtained from known A. radiobacter were tested by the PCR protocols of Haas et al., (1995) which provide a tentative discrimination between pTi and pRi. Strains with fatty acid profiles not consistent with known A. radiobacter strains were stored but not tested for the presence of the plasmid, as it was assumed that rhizogenic A. radiobacter strains were the causal agent of the disorder. Experimental transfer of Agrobacterium pTi, via conjugation, to recipient -Proteobacteria in other genera has been reported (Hooykass et al, 1977, Teyssier-Cuvelle et al, 1998). Hooykaas et al. reported that Rhizobium leguminosarum biovar trifolii transconjugants were able to induce tumours on inoculated host plants. Here we report the isolation, from root mat affected cucumber and tomato crops, of pRi-harbouring -Proteobacteria which do not belong to the Agrobacterium genus. An in vitro hairy root culture assay was modified to act as a rapid host test to determine if these strains could induce typical root mat symptoms on inoculated cucumber cotyledons.
Methods and Results In total over 300 strains isolated over the years from infected crops were tested by two PCR assays (Haas et al, 1995; Weller & Stead, 2002) for the presence of Ri-plasmid (pRi) DNA. Strains positive for both PCRs were further characterised by fatty acid profiling (Stead et al, 1992). In total nine strains were identified which did not belong to the Agrobacterium genus but which did possess pRi DNA. All these strains belong to the -Proteobacteria subgroup and as such are related, in varying degrees, to the Agrobacterium genus. DNA sequencing of the 16S rRNA gene identified five of the strains as Rhizobium, three as Ochrobactrum and one as Sinorhizobium. This sequencing offers the conclusive proof that the strains involved are not Agrobacterium. Although the Agrobacterium Ti-plasmid has been transferred, experimentally, to non-Agrobacterium spp., to the best of our knowledge this is the first time Ri or Ti-plasmid harbouring non-Agrobacterium strains have been isolated from environmental samples.
A cucumber hairy root assay was successfully adapted to provide an in vitro host test. Cucumber cotyledon leaves were cultured on hormone free MS medium. Leaves inoculated with A. radiobacter strains containing a pRi typically developed callus tissue and subsequent roots from the callus. Such roots were generally thick in appearance with irregular, or no, branching (Fig. 1.1), and were similar in appearance to roots in natural outbreaks. Symptoms developed within 2-4 weeks of inoculation. A panel of rhizogenic A. radiobacter strains isolated from affected cucumber crops all induced symptoms, demonstrating the validity of the assay. Roots occasionally grew from negative control cotyledons. However no calli were associated with these leaves and roots were thin and regularly branched, indicating that this was normal regeneration and not the result of a genetic transformation.
CSG 15 (Rev. 6/02) 3 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Figure 1.1. Rooting from cucumber cotyledon leaf inoculated with rhizogenic Agrobacterium radiobacter strain (NCPPB 4042) – 12 days post inoculation.
Using the in vitro assay all nine pRi-harbouring -Proteobacteria strains were shown to have the ability to induce a callus and subsequent transformed roots (Table 1.1). Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) were lower than those observed from rhizogenic A. radiobacter (64%). However individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with the rhizogenic Sinorhizobium (75%). Care must be taken when extrapolating the results from an in vitro assay to those conditions found in a glasshouse. It is not known whether the successful elimination of rhizogenic A. radiobacter from a nursery would allow rhizogenic -Proteobacteria strains to induce symptoms to the same degree.
TABLE 1.1. Characterisation and pathogenicity of pRi-harbouring -Proteobacteria and representative Agrobacterium strains isolated from root mat affected cucumber and tomato crops. Strainsa Identificationb Isolated from Cucumber Hairy Root Culturec CSL 2411 Rhizobium sp. Cucumber Positive (5/11 – 45.5%) CSL 2412 Rhizobium sp. Cucumber Positive (2/14 – 14.3%) CSL 2542 Rhizobium sp. Cucumber Positive (4/11 – 36.4%) CSL 2573 Ochrobactrum sp. Cucumber Positive (8/12 – 66.6%) CSL 2611 Sinorhizobium sp. Cucumber Positive (9/12 – 75%) CSL 2637 Ochrobactrum sp. Cucumber Positive (3/12 – 25%) CSL 3809 Ochrobactrum sp. Tomato Positive (5/11 – 45.5%) CSL 4520 Rhizobium sp. Cucumber Positive (9/13 – 69.2%) CSL 4733 Rhizobium sp. Cucumber Positive (7/13 – 53.8%) All rhizogenic Agrobacterium strains A. radiobacter Cucumber Positive (47/74 – 63.5%) a CSL, plant bacteria collection, Central Science Laboratory. b Identification based on fatty acid profiling and 16S rRNA sequence analysis. c Positive results from total number of discs surviving in culture for at least 14 days
Discussion
The small sample size of isolated rhizogenic -Proteobacteria means it was not possible to determine the full taxonomic range of pRi transfer in root mat affected rhizospheres. The nine strains were, in effect, isolated by accident during a process designed to isolate Agrobacterium strains. As many -Proteobacteria are uncultivable on the media used in this study, many potential transconjugants may not have been isolated. Indeed only cultivable strains were analysed in this report, and as more than 99% of bacteria present in environmental samples are generally considered to be uncultivable we cannot be certain that pRi cannot disseminate outside -Proteobacteria.
All isolated pRi-harbouring -Proteobacteria induced root mat symptoms following in vitro inoculation of cucumber cotyledons. The pathogenicity of such strains, especially in a natural environment, will not be determined solely by the acquisition of pRi. Chromosomal virulence genes, encoding functions such as bacterial attachment to the plant cell or exopolysacchride production, are a necessary component of the Agrobacterium transformation mechanism. Experimental transfer of a pTi to Rhizobium leguminosarum biovar trifolii resulted in this strain becoming pathogenic, though transfer of another pTi to Sinorhizobium meliloti did not result in a pathogenic strain despite vir gene induction and T-DNA
CSG 15 (Rev. 6/02) 4 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
formation. This failure was possibly due to poor attachment of the bacteria to the plant cells or failure of T-DNA transfer from the bacterium and indicates that transfer of pRi may not always result in pathogenic -Proteobacteria strains in root mat rhizospheres.
Although the in vitro test is limited in that leaf, rather than root, tissue is inoculated average transformation rates were higher for rhizogenic A. radiobacter strains than the rates observed from rhizogenic Rhizobium and Ochrobactrum strains. However individual strains from both of these genera, plus the rhizogenic Sinorhizobium strain, showed higher transformation rates, comparable to those seen with individual A. radiobacter strains. This implies that, although a randomly selected pRi-harbouring A. radiobacter strain is more likely to induce extensive symptoms, strains exist outside the Agrobacterium genus which could also be effective pathogens.
During the last year of this project, funding was obtained from Plant Health Division (Defra) to test three of these nine strains in whole plant cucumber host tests. One Ri-plasmid harbouring strains from each of the three genera was tested. Only Rhizobium CSL 2411 was able to induce symptoms on whole plants. This strain induced symptoms as effectively as rhizogenic Agrobacterium NCPPB 4042 control and indicates that efficient root mat pathogens exist, outside of Agrobacterium, though the presence of the Ri-plasmid does not automatically confer pathogenicity on the recipient strain.
The aim of this project was to select biocontrol agent(s) which can control root mat in hydroponic crops. Ri-plasmid transfer to other -Proteobacteria from A. radiobacter complicates this choice. A biocontrol agent that specifically targets A. radiobacter may only lead to root mat being induced by non-Agrobacterium strains harbouring pRi. The numbers and genetic diversity of potential pRi recipients suggests that there is a high probability that many efficient non- Agrobacterium root mat inducers exist. Thus biocontrol agent(s) with a broad range of action / protection may be the best avenue of success. Even if no efficient non-Agrobacterium pathogens exist, rhizogenic -Proteobacteria will play an important epidemiological role in the disease. Avirulent Agrobacterium strains are ubiquitous both on hydroponic cucumber nurseries and in nature, and thus pRi-harbouring non-Agrobacterium species may act as reservoirs of pRi.
Objective 3. To assess the variability of the Agrobacterium Ri-plasmid in root mat associated, and other, strains.
Introduction A real-time PCR assay targeted to the T-DNA of cucumopine pRi (Weller & Stead, 2002) showed that all five rhizogenic A. radiobacter strains isolated from the 1970s root mat outbreak (NCPPB 2655, 2656, 2657, 2659 and 2660), and the majority of root mat associated (RMA) pRi (>80%) isolated from recent outbreaks, possessed the same T-DNA sequence. However some strains from recent outbreaks were isolated which did not possess this sequence but were shown to harbour pRi by the conventional PCR assay (Haas et al., 1995), indicating that other pRi types exist in root mat affected crops. It is possible that RMA pRi from cucumber and tomato differ which may affect the ability of biocontrol agents to control root mat.
Preliminary evidence that different pRi types exist within root mat associated Agrobacterium strains was provided in strains isolated from two neighbouring tomato nurseries. Fifty strains isolated from one nursery, with an exceptionally severe root mat outbreak, possessed Ri-plasmids that reacted with the pRi rol TaqMan PCR assay whilst fourteen strains from the adjoining nursery, with less severe symptoms, possessed pRi which did not. The rol assay is targeted to the T- DNA of the cucumopine pRi type known to induce root mat. The fact that some pRi associated with crops showing root mat symptoms do not possess the same T-DNA indicates that different plasmid types exist. Although these results indicate that the less severe symptoms are associated with the pRi type without the rol sequence, not enough strains from other outbreaks have been isolated to determine whether or not this is significant.
Methods and Results Seventeen root mat associated strains were selected for plasmid profiling (Table 3.1), together with three Agrobacterium reference strains. Root mat associated strains were isolated from affected cucumber and tomato crops in the UK and France in the 1970s and current outbreaks. All but two of these strains harbour cucumopine pRi. The two other strains (CSL 5133 & 5193) possess pRi of an unknown opine type. To avoid complicated plasmid isolation procedures it was decided that the most suitable method was to conduct restriction enzyme analysis of PCR products generated from the T-DNA of these plasmids. Four PCR assays (PCRs A, B, C and D) were developed that each generated 1.5Kb products from different regions of the 16Kb T-DNA region. It was verified that all four PCR products were generated from each of the fifteen strains. Two restriction enzymes (Alu I and Msp I) were obtained which each had several cutting points within these products.
CSG 15 (Rev. 6/02) 5 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
TABLE 3.1. Sources and characteristics of pRi and pTi profiled in this study. Host strain and ID1 Plasmid Source Disease Country Year Isolated rol PCR A. radiobacter NCPPB 2655 pRi Cucumber Root mat UK 1974 + A. radiobacter NCPPB 2656 pRi Cucumber Root mat UK 1974 + A. radiobacter NCPPB 2657 pRi Cucumber Root mat UK 1974 + A. radiobacter NCPPB 2659 pRi Cucumber Root mat UK 1974 + A. radiobacter NCPPB 2660 pRi Cucumber Root mat UK 1974 + A. radiobacter NCPPB 4042 pRi Cucumber Root mat UK 1998 + A. radiobacter NCPPB 4043 pRi Cucumber Root mat UK 1997 + A. radiobacter CSL 5012 pRi Cucumber Root mat France 2002 + A. radiobacter CSL 3276 pRi Tomato Root mat UK 1998 + A. radiobacter NCPPB 4062 pRi Tomato Root mat UK 1998 + A. radiobacter CSL 5083 pRi Tomato Root mat UK 2003 + Rhizobium sp. CSL 2411 pRi Cucumber Root mat UK 1997 + Sinorhizobium sp. CSL 2611 pRi Cucumber Root mat UK 1997 + Ochrobactrum sp. CSL 2573 pRi Cucumber Root mat UK 1997 + Ochrobactrum sp. CSL 3809 pRi Tomato Root mat UK 2000 + A. radiobacter CSL 5133 pRi Tomato Root mat UK 2003 - A. radiobacter CSL 5193 pRi Tomato Root mat UK 2003 - A. tumefaciens C58C1 pTi Cherry Gall USA Unknown - A. tumefaciens NCPPB 2437 pTi Unknown Gall Unknown Unknown - A. rhizogenes NCPPB 2991 pRi Apple Hairy Root Unknown Unknown -
1 Agrobacterium strains identified by fatty acid profiling (Stead et al., 1992); other -Proteobacteria by partial 16S rRNA sequencing (Weller et al., 2004). NCPPB strains are from the National Collection of Plant Pathogenic Bacteria, CSL, Sand Hutton, York, UK; CSL strains from the plant pathogenic bacteria culture collection CSL.
A protocol was developed that led to the successful generation of restriction fragments from these PCR products. Fifteen bacterial strains harbouring cucumopine type pRi all gave PCR products of the expected sizes for all four T-DNA assays. Neither the two other RMA pRi harbouring A. radiobacter strains or any of the three Agrobacterium reference strains produced PCR products from the T-DNA assays. Fragment patterns following restriction of the four PCR products with each of the two restriction enzymes were generally the same for each PCR product / restriction enzyme combination (Fig. 3.1). However the T-DNA C PCR product derived from the NCPPB 2655 A. radiobacter strain gave slightly different banding patterns when cut with both of the restriction enzymes. This result was confirmed by replication.
The T-DNA assay only analyses one of the regions found on the pRi. Therefore a similar experiment was conducted using the virD2 gene, found on all pRi and pTi as the target sequence. A 334 bp product was amplified using a previously published PCR assay and restricted with the same restriction enzymes (Alu I and Msp I). Each of the fifteen strains with the cucumopine pRi, plus two root mat associated pRi which did not react with the rol PCR and three other AgrobacteriumBOX-PCRT-DNA Restriction reference Analysis strains were analysed using this assay. Fragment patterns following restriction of the PCR product with each of the two restriction enzymes were the same for all 15 cucumopine pRi and differed from patterns produced from the PCR products amplified from pRi harboured by two other RMA1 A. radiobacter strains (CSL 5133 and Agrobacterium radiobacter Tomato CSL 3276 .UK 5193) and the A. rhizogenes type strain (NCPPB 2991), and the two pTi harboured by the A. tumefaciens strains C58 and NCPPB 2437 (Fig 3.2). TheseAgrobacterium results indicateradiobacter that theCucumber cucumopine NCPPB pRi are 2659 a unique.UK (1970s) and conserved class of Ri plasmid. Agrobacterium radiobacter Tomato CSL 5083 .UK Discussion Agrobacterium radiobacter Cucumber NCPPB 4042 .UK The results of this study suggestAgrobacterium that one radiobactercucumopine CucumberpRi type survived NCPPB without4043 .UK inducing reported outbreaks of root mat in the UK for around 15 yearsRhizobium prior to thesp. first reportsCucumber of the disease CSL in 2411 hydroponic.UK cucumber crops in 1993. The same pRi has subsequently been responsible, at least partly, for outbreaks of the disease in hydroponic tomato crops in the UK Agrobacterium radiobacter Cucumber NCPPB 2660 .UK (1970s) and cucumber crops in France. We have also shown that this pRi has spread outside the Agrobacterium genus to other α- Proteobacteria during recentSinorhizobium outbreaks, thoughsp. there isCucumber no reason CSL to suppose 2611 that.UK this did not first occur in the 1970s, assuming that this pRi actuallyAgrobacterium originatedradiobacter within AgrobacteriumCucumber . NCPPB 2657 .UK (1970s) FIGURE 3.1. Restriction patternsAgrobacterium of the amplifiedradiobacter T-DNACucumber D region CSL from 5012 15 bacterial.France strains harbouring the cucumopine pRi, after digestion with theOchrobactrum AluI restrictionsp. enzyme Tomato CSL 3809 .UK Ochrobactrum sp. Cucumber CSL 2573 .UK Agrobacterium radiobacter Tomato NCPPB 4062 .UK CSG 15 (Rev. 6/02) Agrobacterium radiobacter Cucumber6 NCPPB 2656 .UK (1970s) Agrobacterium radiobacter Cucumber NCPPB 2655 .UK (1970s) Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
FIGURE 3.2. Restriction patterns of the amplified virD2 region from 17 RMA bacterial strains harbouring pRi and 3 reference strains (NCPPB 2437, NCPPB 2991, C58) after digestion with the MspI restriction enzyme.
virD2 Restriction analysis
Agrobacterium rhizogenes Apple .NCPPB 2991(T) .Unknown Agrobacterium radiobacter Cucumber .NCPPB 4043 .UK Agrobacterium radiobacter Cucumber .NCPPB 4062 .UK Agrobacterium radiobacter Cucumber .NCPPB 2655 .UK (1970s) Agrobacterium radiobacter Cucumber .NCPPB 2656 .UK (1970s) Agrobacterium radiobacter Cucumber .NCPPB 2659 .UK (1970s) Agrobacterium radiobacter Cucumber .NCPPB 2657 .UK (1970s) Agrobacterium radiobacter Tomato .CSL 5083 .UK Ochrobactrum sp. Tomato .CSL 3809 .UK Agrobacterium radiobacter Cucumber .CSL 5012 .France Ochrobactrum sp. Cucumber .CSL 2573 .UK Sinorhizobium sp. Cucumber .CSL 2611 .UK Agrobacterium radiobacter Tomato .CSL 3276 .UK Rhizobium sp. Cucumber .CSL 2411 .UK Agrobacterium radiobacter Cucumber .NCPPB 2660 .UK (1970s) Agrobacterium radiobacter Cucumber .NCPPB 4042 .UK Agrobacterium tumefaciens Cherry .C58 .USA Agrobacterium tumefaciens Unknown .NCPPB 2437(T) .Unknown Agrobacterium radiobacter Tomato .CSL 5133 .UK Agrobacterium radiobacter Tomato .CSL 5193 .UK
1Legends indicate (from left); host bacterial strain; host crop; ID code (NCPPB strains are from the National Collection of Plant Pathogenic Bacteria, CSL, Sand Hutton, York, UK; CSL strains from the plant pathogenic bacteria culture collection CSL, Sand Hutton, York); country of isolation.
Hybridisation studies have shown that the cucumopine NCPPB 2659 pRi is closely related to the mikimopine Ri-plasmid pRi1724 (Moriguchi et al., 2001). Interestingly the mikimopine pRi is also harboured by an Agrobacterium biovar 1 strain (Shiomi et al., 1987), and has caused hairy root disease on melon crops, also a member of the Cucumis genus, in Japan. Although we have shown that the cucumopine pRi is not restricted to Agrobacterium biovar 1 strains these common factors suggest a close evolutionary history at either the bacterial strain or plasmid level.
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Objective 4. To isolate bacteriophages, and also identify other novel classes of control agents, capable of controlling or eliminating pathogenic Agrobacterium sp.
There are two examples which suggest that root mat may be able to be controlled via the application of an agent, or more likely agents, with biological activity against the root mat pathogen. The first is the disappearance of root mat symptoms in soil and straw bed cucumber crops within four or five years of the initial outbreak in the 1970s. The second is the decrease in symptoms seen in an organic tomato crop on the Isle of Wight. This crop, grown in soil, is situated on a nursery with a severe root mat outbreak in hydroponic grown tomatoes. Similar symptoms were initially seen in the organic crop. However between growing seasons the soil beds were amended with green composted waste. A year-on- year reduction in root mat symptoms was observed in the crops until the general disappearance of symptoms. In the hydroponic crops on the same nursery symptoms have remained as severe as before with, if anything, an increase in both the severity and speed in which symptoms have appeared. The most likely explanation for the disappearance of root mat symptoms in both these cases is activity by the existing microflora in the soil reducing the populations of rhizogenic Agrobacterium. There are several possible mechanisms for this reduction which could include organisms having either a direct antagonistic effect or simply outcompeting rhizogenic Agrobacterium strains in the soil. Alternatively populations of organisms could have developed in the rhizosphere which induced a plant defence response that then suppressed populations of Agrobacterium. The removal of affected plants between growing seasons would terminate the release of opines from transformed root tissues and thus remove the favourable niche, at least temporarily, that the infecting Agrobacterium exploited. This potentially gives a window of opportunity for other organisms to exploit. However the nature of this biological control activity is unknown. Millions of organisms exist within even one gram of soil, many of which are unknown to science. It is also possible that different forms of disease suppression developed in different locations. The aim of this project was to somehow reproduce such activity within the rhizosphere of cucumber crops grown in rockwool. Transformation of root cells is irreversible and the resulting root proliferation does not require the presence of rhizogenic Agrobacterium sp. to sustain it. Therefore prevention is the only control method with a chance of success.
4.1.1 Isolation of fluorescent Pseudomonas spp. from cucumber and tomato crops and the development of an in vitro assay to evaluate the potential biocontrol activity of these strains
Introduction
The stated aim of this project was to develop a biocontrol programme that could control root mat in hydroponic crops. It was decided that the first class of potential biocontrol organisms to be isolated would be fluorescent Pseudomonas spp. There were several reasons for this choice which include: . fluorescent Pseudomonas spp. have been shown to provide protection against a range of cucumber diseases (Wei et al., 1996; Natsch et al., 1998; Raupach & Kloepper, 1998; Ongena et al., 1999). These bacteria, along with bacteria with biocontrol activity from other genera such as Bacillus and Stenotrophomonas, are sometimes termed as Plant Growth-Promoting Rhizobacteria (PGPR). . the availability of a commercial product called BioYield (Gustafson), a formulation of two Bacillus strains (B. amyloliquefaciens GB99 and B. subtilis GB122) with known plant growth promoting activity. A sample of this product was kindly supplied by Crompton UK, a subsidiary of Gustafson. This product came with recommended rates of use for both tomatoes and cucumbers and with this is mind it was decided not to attempt to isolate Bacillus sp. direct from UK samples but to assess the potential of this product instead. Methods and Results One-hundred and eighty five samples were collected from a range of UK and French cucumber and tomato nurseries. For each sample the presence of symptoms was recorded, as was the previous history of root mat on the nursery (where known). The presence of Ri-plasmid harbouring bacteria in each sample was assessed by an enriched rol TaqMan PCR assay. Samples were chosen for the likelihood that there was biological control activity already present within the crops from which the sample was taken. Fluorescent Pseudomonas spp. were isolated from these samples via isolation on King’s B (King et al, 1954) or SNA media (Lelliot & Stead, 1987). The main criteria for choosing these samples were: . absence of symptoms at time of sampling or at any time in the previous few years or; . reduction in symptoms from those seen previously. In addition some samples from nurseries with severe root mat outbreaks were also analysed to act as a comparison. In total sixty-six samples were selected from hydroponic cucumber and tomato crops. Soil samples, supporting an organic tomato crop, which had seen a reduction in root mat were also included. The sample types were therefore cucumber or
CSG 15 (Rev. 6/02) 8 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
tomato roots taken from rockwool grown plants, soil collected from the floor of hydroponic nurseries, roots taken from tomato plants in an organic soil bed.
One hundred and eighty one fluorescent Pseudomonas strains were isolated and identified by fatty acid profiling. Phylogenetic analysis of fatty acid data placed the fluorescent Pseudomonas spp. in various closely related clusters of two major divisions, one division consisting of seventy-eight strains (Cluster78) and the other of one hundred and three strains (Cluster103). Within these divisions some clusters comprised strains, from several nurseries, isolated exclusively from one sample type. In each division there were clusters, each consisting of around 30 strains exclusively comprising strains isolated from rockwool grown cucumbers and tomatoes. Forty-one strains were isolated from nurseries with visible symptoms. These strains were distributed unevenly throughout the dendrogram. In Cluster78, twenty-six strains came from known infected nurseries whilst in Cluster103, fifteen strains came from infected nurseries. A cluster within Cluster78 of thirty-four strains contained twenty-two of the strains isolated from the infected nurseries. Ten (of 18 in total) of the strains isolated from roots of organic tomatoes grown in soil were placed in a cluster (within Cluster78) of thirteen strains.
The in vitro pathogenicity test developed previously (Objective 2) was adapted to screen isolated fluorescent Pseudomonas spp. for their ability to prevent cucumber cotyledons being infected by a pRi harbouring A. radiobacter strain. Briefly cotyledons were harvested from one week old seedlings and inoculated with a MS suspension containing a fluorescent Pseudomonas sp. The cotyledons were then incubated on MS medium for 96 hours before being inoculated with the rhizogenic A. radiobacter NCPPB 4042 strain. Cotyledons were then incubated for a further 48 hours before being transferred to MS medium containing the antibiotic cefotaxime. Discs were scored over a period of 3-4 weeks for the presence of callus tissue and subsequent rooting as before.
Thirty one Pseudomonas strains, isolated from hydroponic cucumber crops, were screened using the in vitro assay for the ability to prevent induction of root mat symptoms on cucumber cotyledons. The results of these experiments are summarised in Table 4.1. Sixteen strains appeared to prevent callus and root induction on all surviving cotyledons. Induction of roots was also generally lower on all other cotyledons inoculated with fluorescent Pseudomonas sp. (which did show callus and root induction), than those observed on control cotyledons only inoculated with the rhizogenic A. radiobacter NCPPB 4042 strain. The exceptions to this observation were cotyledons inoculated with Pseudomonas strain CSL 4880. The ability of Bioyield to prevent rooting was also tested using this assay and was shown to have no preventative effect against root mat induction in ten cotyledons tested. However all ten cotyledons inoculated with Bioyield were noticeably healthier than the five negative control cotyledons and the ten positive control cotyledons (solely inoculated with A. radiobacter NCPPB 4042) at 4 weeks post inoculation. This suggests that although Bioyield did have a positive effect on the health of the cotyledons, the mechanisms behind this increase in health did not prevent Agrobacterium infection. This phenomenon was not observed in cotyledons pre-inoculated with any of the Pseudomonas spp. which senesced and died at rates observed previously.
Table 4.1 In vitro screening of fluorescent Pseudomonas spp. and BioYield™ for ability to prevent rooting by rhizogenic A. radiobacter NCPPB 4042 on cucumber cotyledons
CSG 15 (Rev. 6/02) 9 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Treatment ID FAP Cucumber Hairy Root Assay1 Group Pseudomonas sp. CSL 4831 Cluster78 Positive (1/3 – 33%) Pseudomonas sp. CSL 4834 Cluster78 Negative (0/3) Pseudomonas sp. CSL 4856 Cluster78 Negative (0/4) Pseudomonas sp. CSL 4858 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4860 Cluster78 Negative (0/4) Pseudomonas sp. CSL 4836 Cluster78 Negative (0/4) Pseudomonas sp. CSL 4838 Cluster78 Positive (2/5 – 40%) Pseudomonas sp. CSL 4840 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4842 Cluster103 Positive (2/4 – 50%) Pseudomonas sp. CSL 4845 Cluster78 Positive (2/5 – 40%) Pseudomonas sp. CSL 4862 Cluster103 Negative (0/5) Pseudomonas sp. CSL 4864 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4865 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4867 Cluster103 Negative (0/4) Pseudomonas sp. CSL 4868 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4940 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4943 Cluster103 Negative (0/4) Pseudomonas sp. CSL 4854 Cluster103 Negative (0/5) Pseudomonas sp. CSL 4877 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4878 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4879 Cluster103 Positive (2/4 – 50%) Pseudomonas sp. CSL 4880 Cluster78 Positive (4/5 – 80%) Pseudomonas sp. CSL 4881 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4931 Cluster78 Negative (0/5) Pseudomonas sp. CSL 4948 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4951 Cluster103 Negative (0/6) Pseudomonas sp. CSL 4953 Cluster103 Positive (1/5 - 20%) Pseudomonas sp. CSL 4958 Cluster78 Positive (1/5 - 20%) Pseudomonas sp. CSL 4960 Cluster103 Positive (3/5 – 60%) Pseudomonas sp. CSL 4962 Cluster103 Negative (0/5) Pseudomonas sp. CSL 4966 Cluster103 Positive (1/5 - 20%) Bioyield - - Positive (10/10 – 100%) A. radiobacter (only) NCPPB 4042 - Positive (31/35 – 88.5%)
1 Positive results from total number of discs surviving in culture for at least 14 days
Discussion
Fluorescent Pseudomonas were isolated from all but one of the nurseries from which samples were taken for isolation of biological control agents. The presence of such strains on each nursery was not quantified so it is not possible to determine whether there were higher populations on nurseries with no visible symptoms. The cucumopine pRi was detected on the majority of the nurseries from which biocontrol agents were attempted to be isolated. This corresponds CSG 15 (Rev. 6/02) 10 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
with the survey of 1997 where pRi harbouring Agrobacterium sp. were isolated from the majority of UK cucumber nurseries (Weller et al, 2000a). Fluorescent Pseudomonas spp. were isolated from nurseries with no definite symptoms and on which the cucumopine pRi was also detected indicating that the pathogen may be suppressed within crops. Fluorescent Pseudomonas sp. were also isolated from nurseries with definite root mat symptoms though these strains were distributed unevenly on the dendrogram produced from fatty acid data derived from all the isolated strains. Of the two major divisions in the dendrogram (Cluster103 and Cluster78), Cluster78 was divided into two sub-clusters, of thirty-four and forty-three strains respectively (one other strain in Cluster78 did not fall into either of the sub-clusters). In the cluster of thirty-four strains, twenty-six were isolated from nurseries with definite symptoms. If fluorescent Pseudomonas do not reduce root mat within hydroponic cucumber and tomato crops then it might be expected that there should be no difference in the distributions of strains from infected or healthy crops and that one strain from an infected crop be placed every four of the strains isolated from healthy nurseries. One cluster consisting of nearly 75% of strains isolated from nurseries with definite symptoms indicates that although not all fluorescent Pseudomonas strains can suppress the root mat pathogen there may be strains that can.
Fluorescent Pseudomonas spp. placed in both main clusters and isolated from cucumber roots showed the ability to suppress root mat formation in the cucumber cotyledon in vitro assay. This demonstrates that PGPR strains may be able to prevent root mat in the rhizosphere and may have been involved in the suppression of root mat in soil grown crops. However as this test essentially looks at infection of leaf tissues, care must be taken before assuming that these strains could prevent root mat in the rhizosphere of entire plants. It also must not be assumed that Bioyield, which did not prevent root induction in the in vitro assay but which did induce a marked health benefit in the cotyledons, cannot prevent root mat in the rhizosphere as well. This test only give an indication of what might be possible and whole plant tests must be conducted in order to give the true picture.
4.1.2 First STC Glasshouse Trial Introduction Four of the fluorescent Pseudomonas strains (CSL 4836, 4867, 4840 and 4962) which showed ability to prevent root mat induction in the in vitro assay were chosen to be tested in the first large scale glasshouse trial in the final year of the project. This was conducted at the Stockbridge Technology Centre (STC). Two strains from each of the main fatty acid clusters were chosen (four strains in total). Two strain mixture treatments, consisting of one strain from each cluster, were also included giving six fluorescent Pseudomonas in total. Although Bioyield had not shown any preventative effect in the in vitro assay it was decided to use this product as it had shown to have a plant health benefit in this assay and also as it was a commercially available product and thus is much nearer potential use in the UK, than the isolated Pseudomonas spp.
Methods and Results
Cucumber plants (cv. Aviance) were propagated from seed in vermiculite and rockwool propagation cubes. Plants inoculated with Bioyield were germinated in vermiculite containing Bioyield at a rate of 2.4 g L-1, the highest rate recommended for cucumbers by the manufacturers. Sixteen days from sowing groups of eighteen seedlings were inoculated with one of six suspensions containing the Pseudomonas treatments. Seedlings were inoculated with suspensions containing each individual strain plus two suspensions containing equal concentrations of CSL 4836 & 4867 and CSL 4840 & 4962. Suspensions (c. 108 CFU ml-1) were prepared from cultures grown for 24 hr at 28 oC on nutrient agar. Five ml of suspension was applied to the root system of each plant on three separate occasions (Table 4.1). Thirty four days after sowing plants were placed on rockwool slabs (two plants per slab) in a glasshouse in a randomised block design according to treatment. Each plot consisted of three rockwool slabs each containing two plants giving a total of six plants per plot. There were three replicate plots per treatment giving a total of eighteen plants per treatment.
Thirty-six days after sowing, inoculation of all plants in the glasshouse with rhizogenic Agrobacterium NCPPB 4042 commenced. Plants were inoculated with this strain via the irrigation system. One litre of a c. 10 5 CFU ml-1 NCPPB 4042 suspension was prepared from a 24 hr nutrient agar culture (28 oC). This was added to an irrigation tank containing 1000 litres of standard cucumber nutrient solution. It has been shown previously that Agrobacterium can survive in similar nutrient solutions for a period of at least 35 days (S.A. Weller, unpublished data). Over a period of seven days the 1000 litres in this tank was applied when required by the plants. Assuming Agrobacterium cells were distributed equally in the nutrient solution and equal volumes of solution were applied to each plant then 7 x 105 Agrobacterium cells were applied to each plant over 7 days at a rate of 102 CFU ml-1. Plants were trained as in commercial practice. Root mat symptoms were assessed in each treatment at weekly intervals following a scale of 0-5 with zero being no obvious root mat
CSG 15 (Rev. 6/02) 11 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
symptoms and five being extreme root mat symptoms. The four centre plants in each plot were assessed. Cucumber yield in each treatment was measured until the end of the experiment.
Table 4.2. List of treatments and application timings of treatments in 1st STC cucumber trial.
Root mat symptoms were first observed in plants in five of the eight treatments, but not in any of the control plants only inoculated with the rhizogenic Agrobacterium NCPPB 4042 strain, five weeks after the commencement of inoculation with NCPPB 4042. At six weeks symptoms were observed in plants from all treatments and also in plants inoculated solely with NCPPB 4042. At eight weeks the mean scoring of symptoms was appreciably higher in plants solely inoculated with NCPPB 4042 than in plants from any of the other treatments. This trend continued to the end of the experiment with a higher mean incidence of symptoms in the control plants than those observed in any of the treatments (Fig 4.2). The raw data at each sampling point was analysed by ANOVA using GenStat software (Version 6.0). Significant differences in the mean root mat incidence between treatments from those observed in the control plants was Treatment Application Rate Application timing 1 A. radiobacter NCPPB 4042 only - - 2 Bioyield 2.4g L-1 of vermiculite Incorporation into vermiculite pre-sowing 3 Pseudomonas 1 - CSL 4836 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing 4 Pseudomonas 2 - CSL 4867 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing 5 Pseudomonas 1+2 - CSL 4836 + 4867 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing 6 Pseudomonas 3 - CSL 4840 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing 7 Pseudomonas 4 - CSL 4962 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing 8 Pseudomonas 3+4 - CSL 4840 + 4962 5 ml of 108 cfu ml-1 suspension 16, 30 and 44 days after sowing recorded at 9 and 10 weeks (2nd and 9th June) post inoculation (Table 4.3). Duncan’s test was applied to measure where these differences occurred and it was shown that four treatments were significantly different to the mean at nine weeks post inoculation and all treatments were significantly different to the control at ten weeks post inoculation (Table 4.3).
The weight and number of fruit harvested was recorded throughout this experiment. At the end there was no significant difference between any of the treatments for either total number of fruit or average weight of each fruit, indicating that no treatment either significantly increased or decreased yield.
CSG 15 (Rev. 6/02) 12 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Figure 4.2. Suppression of root mat symptoms in cucum ber by PGP R treatments in 1 st ST C trial (Feb-Jun 2004)
2.00
1.80
1.60 Control (Mean)
s CSL 4836 m o
t 1.40 p CSL 4867 m y s
1.20
t CSL 4836 & 4867 a m
t CSL 4840
o 1.00 o r
f CSL 4962 o
t 0.80 n
e CSL 4840 & 4962 m s s
e 0.60 BioYield s s a
n a
e 0.40 M 0.20
0.00
4 4 4 4 4 4 4 4 4 4 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 /2 4 4 4 5 5 5 5 6 6 6 6 /0 /0 /0 /0 /0 /0 /0 /0 /0 /0 /0 4 1 8 5 2 9 6 2 9 6 3 1 2 2 0 1 1 2 0 0 1 2 Date of assessment
Table 4.3. Mean severity of root mat symptoms in cucumber plants on glasshouse trial inoculated with Pseudomonas spp. and BioYield™
Treatment Assessment of mean root mat symptoms (0-5 index) from 5 to 13 weeks post-inoculation 5 6 7 8 9 10 11 13 Weeks Weeks Weeks Weeks Weeks Weeks Weeks Weeks NCPPB 4042 only 0.001 0.25 0.42 0.58 1.17 1.08 1.25 1.83 CSL 4836 0.08 0.25 0.08 0.08 0.17 0.08 0.25 0.58 CSL 4867 0.08 0.25 0.00 0.25 0.25 0.33 0.42 0.67 CSL 4836 & 4867 0.00 0.33 0.17 0.17 0.25 0.17 0.55 0.83 CSL 4840 0.09 0.18 0.00 0.00 0.27 0.10 0.55 1.09 CSL 4962 0.00 0.08 0.00 0.09 0.18 0.27 0.55 1.27 CSL 4840 & 4962 0.08 0.25 0.17 0.25 0.33 0.25 0.42 0.58 BioYield™ 0.17 0.42 0.08 0.17 0.42 0.33 0.83 0.92
1 Means rounded down to two decimal places. Figures in bold represent means significantly different (Duncan’s Test; p < 0.05) to those observed in the control treatment (NCPPB 4042 only).
Discussion
CSG 15 (Rev. 6/02) 13 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Four of the sixteen fluorescent Pseudomonas strains shown to inhibit root mat symptoms in the in vitro test were tested in the first large scale cucumber trial at STC. As mixtures of strains have been shown to provide better disease suppression than individual strains in previous cucumber experiments (Raupach & Kloepper, 1998) it was decided to combine pairs of strains in two of the treatments. To ensure genetic variation between strains the mixtures consisted of one strain from each of the main clusters. The individual strains were also included to ascertain if mixing strains did have a noticeable effect in reducing root mat symptoms. Bioyield was also tested in the experiment even though it could not prevent root mat symptoms in the in vitro test. There were two reasons for this, the first being that although symptoms were not prevented in the in vitro assay there was a marked plant health benefit in Bioyield inoculated cotyledons and the second being that Bioyield is already commercially available (in the USA) and is therefore much closer to potential use in the UK market than the isolated Pseudomonas strains.
Results from this trial were promising. Observed symptom expression did go up and down which can be attributed to variation in recording symptom expression at each sampling interval. Despite the initial appearance of symptoms in all of the treatments, symptom expression was significantly suppressed in four treatments (CSL 4836; CSL 4836 + 4867; CSL 4840; CSL 4962) , when compared to the inoculated control treatment at 9 weeks post inoculation and at 10 weeks post inoculation significant suppression of symptoms was observed in all treatments. After 11 weeks post inoculation significant suppression of root mat symptoms was not observed though the mean incidence of root mat symptoms in the control plants remained higher than the that seen in other treatments for the remainder of the experiment. At the end of the experiment four plants across the experiment had died of fungal infection.
These results suggest that increasing the rhizosphere microbial diversity in rockwool grown cucumbers can suppress the transformation of root tissues by rhizogenic Agrobacterium NCPPB 4042. The last inoculation of the Pseudomonas spp. occurred at the end of Agrobacterium NCPPB 4042 inoculation. Bioyield was only inoculated via the germination vermiculite. Low levels of possible symptom expression was first observed at around 5 weeks after inoculation. Root growth across the slab surface sometimes occurs that is not actual root mat and it is possible that these initial observations were incorrect. At six to eight weeks definite root mat symptoms were observed in the control plants and after this time symptoms showed an upward trend for all but one of the subsequent weeks. Symptom expression in all the treatments remained fairly constant for the first 10 weeks post inoculation and then increased, at different rates, for the remainder of the experiment. As it takes at least 5 weeks for symptom expression to result post inoculation (Weller et al. 2000) these results suggest that the bacterial biocontrol treatments are suppressing significant Agrobacterium infection for a period of two weeks after the end of inoculation. At this time the Agrobacterium then began to be able to transform root tissues effectively leading to the increase in symptoms in the treatments at after 11 weeks post-inoculation. It may be that continued application of Pseudomonas treatments throughout the experiment may have suppressed root mat symptoms further. An increase in Bioyield application could also have suppressed symptom expression. This could be facilitated by adding Bioyield into more of the growth substrate. In solid form rockwool this could be difficult as Bioyield is not supplied as a wettable agent and thus cubes and slabs could not be drenched with this product. However a different, loose, substrate such as composted bark or coir would allow more of the BioYield to be exposed to the developing rhizosphere.
The phenomenon of mixtures of bacterial strains having an enhanced effect was not observed in this experiment. The CSL 4836 & 4867 treatment had a significant effect in both weeks 9 and 10 but this could well have been due to the effect of CSL 4836 on its own which also had a significant effect in both these weeks. Similar results were observed with the other strain combination with the CSL 4840 & 4962 treatment having an effect in week 10, compared with the single applications of each strain which had effects in both weeks 9 & 10. Therefore in this experiment combinations of strains were not shown to induce an enhanced effect. However as only four strains in two combinations were selected for use in this trial we cannot say that a similar effect would be observed for all strain combinations.
4.2 Isolation of Agrobacterium bacteriophages and second STC glasshouse trial
4.2.1 Isolation of Agrobacterium bacteriophages Introduction Bacteriophages are bacterial viruses able to destroy bacterial cells through lysis. Agrobacterium phages have shown a high degree of host specificity, with not one of a panel of nineteen isolated phages having been shown to infect every Agrobacterium strain also from a panel of nineteen (Roslycky et al, 1962). Thus, as for PGPRs, it is likely that mixtures of phages will provide better protection than the application of single strains, especially when considering that resistance
CSG 15 (Rev. 6/02) 14 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
to individual bacteriophage can quickly evolve within bacteria. It is also fair to say that the commercial exploitation of bacteriophages for control of phytopathogens, has not occurred as it is beginning to for bacterial and fungal agents.
Methods and Results
Bacteriophage isolation using rhizogenic Agrobacterium strains isolated from a tomato nursery with a severe root mat outbreak was attempted, using an agar plate overlay technique. A variation of the technique is described fully in section 4.2.2. Run-off water from this nursery was collected and used as the primary source of bacteriophage. Several plaques were generated on lawns of Agrobacterium, indicating the presence of bacteriophages in the run-off water. However, when these strains were isolated and then re-applied to lawns of other pRi harbouring Agrobacterium strains also isolated from the same nursery, plaque formation was not induced.
Discussion These results indicate that bacteriophages, able to infect cucumopine pRi harbouring Agrobacterium strains, are highly specific and unable to infect all pathogenic Agrobacterium strains present on a single nursery. It does however indicate that bacteriophages do exist on infected nurseries and may indicate that a panel of bacteriophages, applied in excess may have the potential to control root mat. However the presence of non-Agrobacterium strains which harbour the cucumopine pRi and can induce symptoms on whole plants mean that it may be very difficult to obtain a panel of phages able to cover all potential pathogenic strains. This is further complicated by the fact that we do not know the entire host range of the cucumopine pRi.
4.2.2 Second STC Glasshouse Trial
Introduction With the first trial exclusively restricted to the testing of PGPR strains, in the second trial the ability of a variety of other organisms, compounds, and products to suppress root mat was evaluated, along with the fluorescent Pseudomonas strain shown to cause the greatest disease suppression in the first glasshouse trial. In the second trial the following agents were evaluated, Agrobacterium bacteriophages, β-Aminobutyric acid - a known inducer of plant defence mechanisms, a product (Rhizotonic) claimed to promote rhizosphere health; Pasteurised soil. The reasons behind choosing these organisms and products are described below.
Bacteriophages - although the isolation of non-Agrobacterium sp. harbouring the cucumopine pRi suggests that they would not be able to be used as a sole means of control we have included their use in this trial to establish whether there is potential benefit in including them as part of an integrated biocontrol programme.
Plant defence mechanism inducer - the role of compounds such as salicylic acid in inducing a plant defence response such as systemic acquired resistance (SAR) is well known. Commercial exploitation of such compounds is beginning to occur with the compound acibenzolar-S-methyl (BTH) being marketed in the EU as Bion (Novartis). We were unable to obtain this product but in order to establish the principle of use of such a compound in the control of root mat we decided to use -amino butyric acid (BABA) a compound known to induce a resistance against a range of plant pathogens (Cohen, 2002). In order to measure the effects of this compound on its own, and as part of a integrated control strategy we combined BABA with bacteriophages and the fluorescent Pseudomonas sp. CSL 4836, which showed suppression of root mat symptoms in the first STC trial, in two of the three BABA treatments.
Others - products and compounds are available which are stated to improve the health of the rhizosphere in hydroponic production. One of these products (Rhizotonic, Canna) was selected and also included in the treatment. In the sterile rockwool environment this product, as others, state that rhizosphere health is promoted with protection against several plant pathogens. In a similar treatment pasteurised soil was added to the tops of rockwool cubes. Soil that has been pasteurised is free from fungal pathogens but retains thermotolerant bacteria, such as Bacillus species, which may be able to colonise the rockwool and create a rhizosphere environment less favourable to Agrobacterium. If this treatment was found to be effective it is possible that could be applied by growers, for example as a growth promoting material, and would not require registration as a pesticide.
CSG 15 (Rev. 6/02) 15 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Methods and Results Preparation of Agrobacterium bacteriophage suspensions A run-off sample was obtained from the nursery from which the A. radiobacter NCPPB 4042 strain was originally isolated. A quantity of this sample (20 ml) was filter sterilised though a 0.2 μm filter. 0.5 ml of this filtered run-off was added to 0.2 ml of an overnight Agrobacterium NCPPB 4042 culture and placed in 20 ml of fresh nutrient broth (NB). This suspension was incubated overnight in a shaking incubator (28 oC; 50 rpm). To aid bacteriophage attachment to
Agrobacterium cells all media during preparation contained 10 mM CaCl2 and 10 mM MgSO4. The run-off / NCPPB 4042 overnight culture was centrifuged for 15 min at 3, 600 rpm and the supernatant removed and filter sterilised. 0.2 ml of supernatant was added to 0.2 ml of a fresh NCPB 4042 NB overnight culture and allowed to stand for 1 hour at room temperature. 4.5 ml of 0.7 % Nutrient Agar (NA) was then added and the resulting suspension was poured over standard nutrient agar plates (1.5 % agar). Twelve plates were prepared in this manner and incubated overnight. Plates showing plaques were harvested by removing the soft agar with spreaders following the addition of 10 ml of ice-cold TM buffer. The resulting suspensions were bulked and filter Pasteurised. This procedure was repeated twice to produce a more concentrated suspension of phage. Final volumes of 150 ml was produced for each application. 5 mls of the resulting suspension was injected around the crown of each plant, immediately prior to the first inoculation of rhizogenic Agrobacterium and immediately after the end of the inoculation period.
Table 4.4. List of treatments and application timings of treatments in 2nd STC cucumber trial.
Treatment Application Rate Application timing 1 A. radiobacter NCPPB 4042 only 7 x 106 cfu per plant 29-36 days after sowing 2 Pseudomonas CSL 4836 5 ml of 108 cfu ml-1 22, 29, and 36 days after sowing 3 Rhizotonic 250 ml per plant 15, 29, 36, 43 days after sowing 4 Phage 5 mls of phage suspension 29 and 36 days after sowing 5 BABA+ Phage 17 ml of 50 g ml-1 suspension (BABA)1 22, 29, 36 and 43 days after sowing1 6 BABA 17 ml of 50 g ml-1 suspension 22, 29, 36 and 43 days after sowing 7 BABA + Pseud 4836 17 ml of 50 g ml-1 suspension (BABA)1 22, 29, 36 and 43 days after sowing1 8 Pasteurised soil 50 g per plant 22 days after sowing Cucumber plants (cv. Aviance) were propagated from seed in vermiculite and trained as in the first trial. Treatments were applied as in Table 4.4. To increase the stringency of the trial there was a 10-fold increase in the amount of NCPPB 4042 applied to each plant, when compared with the first experiment. Symptoms were recorded as in the first trial. However half way through the trial all 6 plants in each plot were assessed for symptoms as many plants within the trial had started to die of a fungal infection, Mycospharella stem rot.
Means of root mat incidence were only determined from viable plants. No significant differences from symptom expression in the inoculated control plants, at any stage of the experiment, were observed in any of the treatments (Duncan’s test; p < 0.5) (Table 4.5). At the end of the trial the BABA + phage treatment showed the highest level of disease expression, followed by CSL 4836 and then the inoculated control. Symptoms in the Pasteurised soil treatment were the first to appear and progressed steadily throughout the experiment (Figure 4.3). Three treatments, BABA, phage and rhizotonic showed less mean incidence when compared to the inoculated control. As in the first trial the Ri-plasmid was detected in samples taken from all plots at the end of the experiment.
CSG 15 (Rev. 6/02) 16 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
Table 4.5. Mean severity of root mat symptoms in cucumber plants on second STC glasshouse trial.
Treatment Assessment of mean root mat symptoms (0-5 index) from 6 to 12 weeks post-inoculation 6 Weeks 8 Weeks 9 Weeks 10 Weeks 11 Weeks 12 Weeks NCPPB 4042 Only 0.00 0.44 0.44 0.67 1.09 1.27 CSL 4836 0.00 0.08 0.53 0.56 1.07 1.40 Rhizotonic 0.00 0.17 0.28 0.46 0.55 0.55 Phage 0.00 0.08 0.06 0.14 0.25 0.34 BABA+ Phage 0.00 0.17 1.12 1.35 1.53 1.62 BABA 0.00 0.00 0.00 0.17 0.17 0.33 BABA+ CSL 4836 0.00 0.00 0.39 0.43 0.71 1.07 Pasteurised Soil 0.33 0.75 0.88 1.06 1.14 1.21
Figure 4.3 Suppression of root mat symptom s in cucumber by several treatments in 2nd ST C T rial (Aug-Dec 2004)
1.8 Inoc control 1.6 Pseud 4836 1.4 Rhizo Phage
t 1.2 a BABA+Phage m
t o
o BABA r 1
f o
BABA+4836 e c n e 0.8 S. Soil d i c n i
n 0.6 a e M 0.4
0.2
0 06/10/2004 13/10/2004 20/10/2004 27/10/2004 03/11/2004 10/11/2004 17/11/2004 24/11/2004 01/12/2004 Date of assessm ent
Discussion
The results of this trial were less encouraging than those achieved in the first STC trial. At the end of the experiment 29% of the plants had died (of Mycospharella stem rot), unlike in the first trial where less than 3% of plants died of fungal infection before the end of the experiment. In some of the treatments notably the single phage treatment this figure was higher (56%) and this may have affected the results, possibly by increasing or decreasing the mean observation of root mat incidence. This undoubtedly affected the variance used in the statistical calculations and led to no significant difference in root mat incidence being observed between any of the treatments and the inoculated control plants. No control measures for fungal infection were used due to the biocontrol nature of the project.
The fluorescent Pseudomonas CSL 4836 strain, which showed good suppression of root mat symptoms in the first trial, in this instance failed to control root mat symptoms when compared to the inoculated control. The 10-fold increase in the concentration of NCPPB 4042 may have affected the ability of this strain to prevent root mat symptoms, though symptoms in the inoculated control were not higher in this experiment when compared to those observed in the first experiment, not taking into account the number of inoculated control plants that died in the second experiment (38%) CSG 15 (Rev. 6/02) 17 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
when compared to the first experiment (0%). Of the CSL 4836 inoculated plants 18% died before the end of the second experiment, compared with 0% in the first.
The treatments that showed the most promise in the second trial, though which did not induce a significant difference in mean root mat incidence from the inoculated NCPPB 4042 control, were phage, Rhizotonic® and BABA. Of these BABA showed the most effect with no symptoms being observed until 10 weeks after inoculation, compared with 8 weeks in the inoculated control. BABA was applied to plants at seven day intervals from 22 to 42 days after sowing. NCPPB 4042 was inoculated onto plants from 29-36 days after sowing. Therefore the period of BABA inoculation, one week either side of the NCPPB 4042 inoculation, may have induced a defence response within cucumber that prevented or suppressed root mat infection at around this time. However this response was unable to eradicate the pathogen from the rhizosphere and when the application of BABA ended infection was allowed to occur with symptoms resulting 10 weeks later. This suggests that a continued application of BABA throughout the course of the trial may have resulted in continued suppression of symptoms.
When BABA was combined with either CSL 4836 or phage no noticeable suppression of symptoms, when compared with NCPPB 4042 plants was observed, suggesting if there was a defence response induced by the single BABA treatment then this was negated by the application of fluorescent Pseudomonas CSL 4836 or phage. There is no obvious explanation for this unless the defence response affected the biocontrol application, i.e. CSL 4836, more the NCPPB 4042 cells allowing the rhizogenic Agrobacterium to induce symptoms before those observed in the single BABA treatment when the defence response was focused solely on NCPPB 4042.
The single phage treatment also appeared to have a good effect at suppressing symptoms, even though more than half the treated plants had died by the end of the experiment. As viruses, phage would seem to have a good chance of success as a biocontrol agent as, if infection of bacterial cells is successful then phage will become self-propagating and the lysis of individual infected bacterial cells results in the release of more quantities of phage into the rhizosphere, thereby increasing the population of phage. However there are notes of caution. In the second year of the project bacteriophage isolation using rhizogenic Agrobacterium strains isolated from a tomato nursery with a severe root mat outbreak was attempted, using an agar plate overlay technique. Several plaques were generated on lawns of Agrobacterium, indicating the presence of bacteriophages. However isolation of these phages did not result in plaques being generated on lawns of other Agrobacterium sp. from the same nursery, indicating the high specificity of phages. This suggests that for every nursery a panel of phage would have to be isolated to combat all Agrobacterium sp. present on that site. However if other bacterial species harbour the Ri-plasmid, and are able to induce symptoms as effectively as Agrobacterium sp. (like Rhizobium CSL 2411), then it is likely that a panel of phages may need to be isolated for each bacterial strain known to be able to harbour the Ri-plasmid. As we do not know the range of bacterial species able to harbour the Ri-plasmid then phage may not be a practical means of control. If however rhizogenic Agrobacterium are the primary pathogen, and other rhizogenic strains do not exert as big an effect, then phage may be a useful component of an integrated control strategy. There is currently an increase in interest in the use of phage in human, animal and plant health. In all these areas panels of phage have been isolated and specificity of these panels is reviewed regularly. Interest is largely determined by the promise of low cost for approval and registration.
Rhizotonic® was the other treatment which appeared to suppress symptoms. This product is marketed as a “stress reliever” that is able to improve the health of the root system. Whilst the scientific basis behind this claim is not stated it is certainly true, as a generalisation, that stressed crops are more susceptible to Agrobacterium infection especially crops susceptible to crown gall induced by Agrobacterium sp. harbouring Ti-plasmids (S Weller, personal observation). Such a product may well be useful as part of a integrated control strategy if a suitable combination of treatments could be devised.
4.3 Evaluation of organic growth substrates for inherent ability to suppress root mat symptoms
There have been reports and circumstantial evidence that various growth substrates such as coir fibres, hinoki bark or certain composts may possess (or induce) anti-pathogen activity and therefore suppress pathogens which, in rockwool, are not suppressed. A third large scale glasshouse trial was set up at STC to evaluate if three organic growth substrates could suppress root mat symptoms. Instead of using cucumbers, as in the previous two experiments, in this experiment tomatoes
CSG 15 (Rev. 6/02) 18 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
were used. Individual tomato plants are kept in production for the entire growing season, unlike cucumbers which can be replaced. Thus in tomatoes it is very important that root mat be suppressed, as there is no opportunity to replace affected plants once infection has occurred.
Materials and Methods Three growth substrates were selected for evaluation. These were: 1. Melcourt Growbark - this product consists of matured European pine bark. Plants were germinated in Melcourt Sylvamix potting mixture, a peat free growing medium. 2. Wessex Cocopeat - this product consists of coir pith and by-product of the coconut fibre extraction process in Sri Lanka. Like rockwool it has a high water holding capacity and thus is ideal for hydroponic growth systems. Plants were germinated in a mixture of 50% cocopeat / 50% compost. This product was kindly supplied by Wessex Horticultural Products. 3. Canna Coir- another coir product with the addition of the fungus Trichoderma, which is known to protect plants against certain soil-borne diseases. Plants were germinated in a mixture of 50% Canna Coir / 50% compost.
Cherry tomato plants of a variety that has shown severe root mat symptoms (cv. Conchita) were propagated from seed as stated above, with rockwool grown plants being germinated in vermiculite as per normal commercial practice. After germination plants were propagated in pots containing the requisite substrate, with rockwool grown plants being propagated in rockwool cubes. Four weeks after sowing plants were placed into slabs of the requisite growth substrate (2 plants per slab, two slabs per plot) with rockwool grown plants being placed onto slabs. Each treatment consisted of four replicate plots giving sixteen plants in total. After planting plants were inoculated as before via the irrigation system. A virulent Ri-plasmid harbouring Agrobacterium sp. isolated from a tomato nursery with a chronic root mat outbreak was used in this trial. Each plant received approx. 7 x 106 CFU over the week in which inoculation took place. Plants were trained as in commercial practice. Root mat symptoms were assessed in each treatment at weekly intervals following a scale of 0-5 with zero being no obvious root mat symptoms being zero and five being extreme root mat symptoms. Yields were not assessed in this trial.
Symptoms first appeared in rockwool grown plants 4 weeks after inoculation. At 5 weeks symptoms had appeared in each of the other treatments and over the next 2-3 weeks, symptoms in the three organic substrates overtook those observed in the rockwool grown plants (Figure 4.4)
Figure 4.4 Incidence of root mat symptoms in tomatoes grown in four different growth substrates
3.5
Bark 3
Canna Coir 2.5 t a m
Rockwool t o
o 2 r
f o
e
c Wessex Coir n e
d 1.5 i c n i
n a e 1 M
0.5
0 02/06/2004 09/06/2004 16/06/2004 23/06/2004 30/06/2004 07/07/2004 Date of assessm ent
Discussion
CSG 15 (Rev. 6/02) 19 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
These results indicate that the none of the three growth substrates used possessed any ability to significantly reduce incidence of root mat. As each substrate comes in a loose form, unlike moulded rockwool slabs, there is the potential for using each substrate as a base for mixing in a biocontrol agent. However on their own there appears to be no value in growers using such substrates in the expectation that root mat symptoms will be reduced. The fungal agent Trichoderma, known to have biological control abilities against some plant pathogens and present in Canna Coir, also appeared to have no effect against the root mat pathogen.
Objective 5 – To monitor the survival of Agrobacterium and the Ri-plasmid
5.1 Presence of rhizogenic Agrobacterium sp. on nursery weeds and other crops Ninety-one samples from crops other than cucumber and tomato, along with weed species and soil were taken from nine nurseries. The samples included hydroponic aubergines, lettuces and peppers; pot grown basil, parsley, pyracantha, poinsettia and fuchsia; and various weed samples. The samples were enriched in the Agrobacterium sp. semi-selective Medium 1A for 72 hours and then an aliquot of the resulting suspension was tested for the root mat associated Ri-plasmid using the rol TaqMan PCR assay. Positives were only detected from samples at root mat affected tomato and cucumber nurseries and are summarised in Table 5.1. However these included many weed species both inside and outside affected houses. Excessive root proliferation was not noted with these samples which suggests that weed species can act as reservoirs of rhizogenic bacterial strains. However as some soil samples also tested positive for the pathogen it may be that the pathogen resides in soil and the presence of roots is incidental.
In addition to these weed species some pot grown parsley plants also tested positive for the root mat pathogen. These plants were grown in glasshouses on the same site as a hydroponic tomato nursery with a root mat outbreak ongoing at the time of sampling. Although no root proliferation was noted in the parsley rhizosphere, these results illustrate the ubiquitous nature of the organism and its ability to survive on other crop types and environments and may explain a) why it is so difficult to eradicate and b) its spread across much of the UK.
Table 5.1 Protected crops and weed species, from three hydroponic nurseries, which tested positive for Ri-plasmid harbouring bacterial species.
Sample Nursery Inside / Outside Groundsel Hydroponic tomato nursery Outside Chickweed Hydroponic tomato nursery Outside Dandelion Hydroponic tomato nursery Outside Grass Hydroponic tomato nursery Outside Volunteer tomato Hydroponic tomato nursery Outside Unknown weed sp. Hydroponic tomato nursery Outside Unknown weed sp. Hydroponic tomato nursery Outside Unknown weed sp. Hydroponic tomato nursery Outside Soil Hydroponic tomato nursery Outside Volunteer tomato Hydroponic tomato nursery Inside Potted parsley plant Hydroponic tomato nursery Inside Potted parsley plant Hydroponic tomato nursery Inside Plantain Hydroponic tomato nursery Inside Dock Hydroponic tomato nursery Inside Grass Hydroponic tomato nursery Inside Sowthistle Hydroponic tomato nursery Inside Volunteer tomato Hydroponic tomato nursery Inside Soil Hydroponic tomato nursery Outside Grass Hydroponic cucumber nursery Inside Dandelion Hydroponic cucumber nursery Inside Soil Hydroponic cucumber nursery Inside
CSG 15 (Rev. 6/02) 20 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
5.2 Survival of rhizogenic Agrobacterium sp. and Ri-plasmid DNA on nursery substrates The four growth substrates evaluated for any inherent ability to suppress root mat in tomatoes were tested to see if Agrobacterium strains and Ri-plasmid DNA could survive, in the absence of a developing rhizosphere, for a period of three weeks. Surviving Agrobacterium strains in a substrate re-used in another growing season could clearly be a source of re-infection, especially as in steam sterilising large quantities of slabs it is very likely that cool spots under tarpaulins (used to trap the steam) could allow the survival of strains. It is also possible that Ri-plasmid DNA, which is likely to survive from strains that have been killed in the steaming process, could be acquired by avirulent Agrobacterium sp. ingressing into these slabs on the resumption of propagation. There is the theoretical possibility that such strains could then become pathogenic.
Methods and Results
Five grams of each of the substrates (replicated) was acquired and put into sterile universal tubes. To these samples 0.5 ml of a 108 CFU Agrobacterium NCPPB 4042 suspension was added, the tubes were closed and vortexed. One set of replicates was dried overnight in an oven (80 oC). Both sets of tubes were then stored at room temperature for 20 days. At 21 days DNA was extracted from 0.2 g of dried substrate using a DNA extraction kit (Wizard Magnetic DNA Purification System for Food, Promega Corporation. Madison, WI). This extract was used as template in the rol real-time PCR assay to determine of Ri-plasmid DNA had survived in the dried tubes. At the same time 0.2 g of substrate was removed from each of the non-dried tubes and added to 1 ml of sterile phosphate buffer. One hundred l of the resulting suspensions were plated onto NA plates and incubated at 28 oC for 24 hours.
Ri-plasmid DNA was detected from the rockwool and coir substrates but not bark. Bacterial loads were equivalent in coir and bark but much higher in rockwool (Table 5.2)
Table 5.2 Evaluation of bacterial load and presence of Ri-plasmid DNA in four growth substrates, 21 days after inoculation with a pRi harbouring Agrobacterium suspension.
Substrate Estimate of No. of Agrobacterium rol PCR result on DNA extracted from colonies per ml of sample suspension dried substrate Melcourt Growbark 1000-10000 - Canna Coir 1000-10000 + Wessex Cocopeat 1000-10000 + Grodan Rockwool 10 000+ +
Discussion These results are interesting as they suggest that composted bark is unable to retain Ri-plasmid DNA, after the substrate has been dried, unlike the coir substrates and rockwool. In the non-dried substrates there also appeared to be less retention of the pathogen in the organic substrates than in rockwool. These are interesting results but in the context of STC Trial 3, which showed that these substrates where unable to prevent root mat in tomatoes, they would seem to be academically interesting rather than having any practical importance.
Objective 6 – Technology Transfer
Results of this project have been passed onto growers at the earliest possible opportunity, through the consultants involved with the project (Derek Hargreaves and Dr Phil Morley) and also via ADAS and STC. An article describing the findings of the project has been written for Defra Plant It! Magazine. Root mat has become a chronic problem on some tomato nurseries. The findings of this project have already been discussed with a major tomato nursery and a project proposal has already been submitted to the Horticultural Development Council (HDC) to extend the biological control findings of this work. HDC have agreed to fast track this proposal with a view to the project commencing on the 1st April 2005.
CSG 15 (Rev. 6/02) 21 Project Improved control of novel Agrobacterium induced DEFRA HH2308SPC title diseases in hydroponic crops through risk assessment project code and biological control.
One scientific paper has been published (Weller SA, Stead DE, Young JPW. 2004. Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other α-Proteobacteria in cucumber and tomato crops affected by root mat. Applied and Environmental Microbiology 70: 2779-2785) and a talk entitled “The cucumopine Ri plasmid and root mat in cucumbers and tomatoes” was presented to the 25th Crown Gall Conference at the University of Illinois in August 2004. Two other scientific papers relating to work conducted in this project are currently in preparation.
References
Cohen YR. 2002. β-Aminobutyric acid-induced resistance against plant pathogens. Plant Disease 86: 448-457. Haas JH, Moore LW, Ream W, Manulis S. 1995. Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Applied and Environmental Microbiology 61: 2879-2884. Hooykaas PJJ, Klapwijk PM, Nuti MP, Schilperoort RA, Rörsch A. 1977. Transfer of the Agrobacterium tumefaciens TI plasmid to avirulent agrobacteria and to Rhizobium ex planta. Journal of General Microbiology 98:477-484. King EO, Ward MK, Raney DE. 1954. Two single media for the demonstration of pyocyanin and fluoroscein. Journal of Laboratory and Clinical Medicine 44: 301-307. Lelliot RA, Stead DE. 1987. Methods for the diagnosis of bacterial diseases of plants. Blackwell Scientific Publications, Oxford, UK. Moriguchi K, Maeda Y, Satou M, Hardayani NSN, Kataoka M, Tanaka N, Yoshida K. 2001. The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in Rhizobiaceae. Journal of Molecular Biology 307: 771-784. Natsch A, Keel C, Hebecker N, Laasik E, Defago G. 1998. Impact of Pseudomonas flourescens strain CHA0 and a derative with improved biocontrol activity on the culturable resident bacterial community on cucumber roots. FEMS Microbiology Ecology 27: 365-380. Ongena M, Daayf F, Jacques P, Thonart P, Benhamou N, Paulitz TC, Cornelis P, Koedam N, Belanger RR. 1999. Protection against Pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and anitbiosis. Plant Pathology 48: 66-76. Raupach GS, Kloepper, JW. 1998. Mixtures of Plant Growth Promoting Rhizobacteria enhance biological control of multiple cucumber pathogens. Phytopathology 88: 1158-1164. Roslycky EB, Allen ON, McCoyE. 1962. Phages for Agrobacterium radiobacter with reference to host range. Canadian Journal of Microbiology 8: 71-78. Shiomi T, Shirakawa T, Takeuchi, Oizumi T, Uematsu S. 1987. Hairy root of melon caused by Agrobacterium rhizogenes biovar 1. Annals of the Phytopathological Society of Japan. 53: 454-459. Stead DE, Sellwood JE, Wilson J, Viney I. 1992. Evaluation of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria. Journal of Applied Bacteriology 72: 315-321. Teyssier-Cuvelle S, Mougel C, Nesme X. 1999. Direct conjugal transfers of Ti-plasmid to soil microflora. Molecular Ecology 8: 1273-1284. Wei G, Kloepper JW, Tuzun S. 1995. Induced systemic resistance to cucumber diseases and increased plant growth by plant growth promoting rhizobacteria. Phytopathology 86: 221-224. Weller SA, Stead DE, O’Neill, TM, Hargreaves D, McPherson G.M. 2000a. Rhizogenic Agrobacterium biovar 1 strains and cucumber root mat in the UK. Plant Pathology 49: 43-50. Weller SA, Stead DE, O’Neill TM, Morley PS. 2000b. Root mat of tomato caused by rhizogenic strains of Agrobacterium biovar 1 in the UK. Plant Pathology 49: 799. Weller SA, Stead DE. 2002. Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR. Journal of Applied Microbiology. 92: 118-126. Weller SA, Stead DE, Young JPW. 2004. Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other α-Proteobacteria in cucumber and tomato crops affected by root mat. Applied and Environmental Microbiology 70: 2779-2785.
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