Biocontrol compositions
11690374 · 2023-07-04
Assignee
Inventors
Cpc classification
A01N25/00
HUMAN NECESSITIES
International classification
A01N25/00
HUMAN NECESSITIES
Abstract
The invention provides isolated Erwinia persicina strains with activity against: a) at least one Xanthomonas species, and/or b) at least one Brassicaceae pathogen. In particular the invention provides the isolated E. persicina strains deposited as DSM 32302, DSM 32304, DSM 32305 and DSM 32303. The invention provides compositions comprising one or more strains of the invention. The invention also provides methods of use of one or more strains or compositions of the inventions to control plant pathogens, particularly Xanthomonas campestris pv. campestris.
Claims
1. A method for controlling at least one Xanthomonas species, the method comprising contacting at least one Xanthomonas species with an isolated Erwininis persicina strain with activity against at least one Xanthomonas species.
2. A method for controlling at least one Xanthomonas species on or in a plant, plant part, seed, or soil comprising applying at least one of: i) an isolated Erwininis persicina strain with activity against at least one Xanthomonas species, and ii) a composition comprising an isolated Erwininis persicina strain with activity against at least one Xanthomonas species, to the plant, plant part, seed, or soil.
3. The method of claim 2 in which the strain or composition has a direct effect to control the at least one Xanthomonas species.
4. The method of claim 2 in which the strain or composition affects induced systemic resistance in the plant, plant part, or seed, to control the at least one Xanthomonas species.
5. The method of claim 1 in which the at least one Xanthomonas species is at least one of: a) Xanthomonas campestris, b) a Xanthomonas species that causes black rot, and c) Xanthomonas campestris pv. campestris.
6. The method of claim 2 in which the plant, plant part, or seed is at least one of: a) from a Brassicaceae plant, b) from a Brassicaceae plant of the Brassica genus, c) from B. oleracea, and d) from B. rapa.
7. The method of claim 2 in which the at least one strain or composition is applied to a seed hole before planting a seed, and the seed then contacts the at least one strain or composition when it is planted in the seed hole.
8. The method of claim 2 in which the at least one strain or composition is applied to a seed of a plant before planting.
9. The method of claim 8 in which the at least one strain or composition is applied to the seed: a) in the form of a seed coat, or b) by bio-priming.
10. A method for inoculating a plant, or plant part against at least one Xanthamoos species comprising contacting the plant, or plant part, with at least one of: i) an isolated Erwininis persicina strain with activity against at least one Xanthomonas species, and ii) a composition comprising an isolated Erwininis persicina strain with activity against at least one Xanthomonas species.
11. The method of claim 10 in which the plant part is a seed.
12. The method of claim 11 in which the seed is coated or bio-primed with at least one of: i) the isolated Erwininis persicina strain with activity against at least one Xanthomonas species, and ii) the composition comprising an isolated Erwininis persicina strain with a ctivity against at least one Xanthomonas species.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The invention will now be described with reference to the Figures in the accompany drawings in which:
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(16) ) was treated with bacteriological peptone water. Growth room conditions cycled from 25° C. light for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The error bars indicate the LSD (5%) for comparison of the positive control against isolates 90, 1774 and 1860 (a) and isolate 76 (b), and for comparisons between isolates 90, 1774 and 1860 (c), isolate 76 and the other isolates (d) and the different rates of isolate 76 (e).
(17) ; 30 replicates) was treated with bacteriological peptone water. The error bars indicate the LSD (5%) for comparison of treatments with 10 versus 30 replicates (a) and 10 versus 10 replicates (b).
(18) ; 30 replicates) and negative (
; 20 replicates) controls (with and without Xcc, respectively) was treated with bacteriological peptone water. The error bars indicate the LSD (5%) for comparison of treatments with 20 versus 30 replicates (a), 10 versus 30 replicates (b) and 10 versus 10 replicates (c).
(19) ; 30 replicates) was treated with bacteriological peptone water. Growth room conditions cycled from 25° C. light for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The error bars indicate the LSD (5%) for comparison of treatments with 30 versus 30 replicates (a), 15 versus 30 replicates (b) and 15 versus 15 replicates (c).
(20) , 30 replicates) and negative (
; 15 replicates) controls (with and without Xcc, respectively) was treated with bacteriological peptone water. Growth room conditions cycled from 25° C. light for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The error bars indicate the LSD (5%) for comparison of treatments with 30 versus 30 replicates (a), 15 versus 30 replicates (b) and 15 versus 15 replicates (c).
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(28) , respectively) at target rates of 3×10.sup.7 (low), 3×10.sup.8 (medium) and 3×10.sup.9 (high) CFU/g seed, to seed artificially inoculated with Xanthomonas campestris pv. campestris (Xcc) isolate ICMP 21080 (15 replicates of each). Seed for the positive (Xcc) controls was treated with the seed coating (
) and standard seed treatment (
) without BCA (30 replicates of each). Growth room conditions cycled from 25° C. light for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The error bars indicate the LSD (5%) for comparison of treatments with 30 versus 30 replicates (a), 15 versus 30 replicates (b) and 15 versus 15 replicates (c).
(29) , respectively) at target rates of 3×10.sup.7 (low), 3×10.sup.8 (medium) and 3×10.sup.9 (high) CFU/g seed (15 replicates of each). Seed for the positive (Xcc) controls was treated with the seed coating (
) and standard seed treatment (
) without BCA (30 replicates of each). Growth room conditions cycled from 25° C. light for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The error bars indicate the LSD (5%) for comparison of treatments with 30 versus 30 replicates (a), 15 versus 30 replicates (b) and 15 versus 15 replicates (c).
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(38) ) was treated with bacteriological peptone water. The error bar to the right of the positive control data points indicates the LSD (5%) for that timepoint.
(39) ) was treated with bacteriological peptone water and the spray without BCA was applied to transplants. The error bar to the right of the positive control data points indicates the LSD (5%) for that timepoint.
EXAMPLES
(40) The following non-limiting Examples are provided to illustrate the present invention and in no way limit the scope thereof.
Example 1: Process for Isolation of Erwinia persicina
(41) As part of a search for novel biocontrol agents (BCAs) of pests and diseases of brassicas, microbes were isolated from 47 seed lots of 10 brassica plant types; the vegetables: broccoli, cabbage, cauliflower, raddish, kohlrabi and pak choi, and the forage plants: kale, turnip, rape and swede.
(42) Seeds from each seed lot (stored in moisture-proof containers at 4° C.) were randomly divided into two groups of approximately equal numbers. One of these groups was further subdivided in half or thirds for surface sterilization with 1, 2 and/or 3% NaOCl. The seeds were surface-sterilized in 70% (v/v) ethanol for 30 s followed by shaking at 200 rpm for 2 min in 1, 2 or 3% NaOCl with 0.01% (v/v) Tween 20. They were then rinsed three times with sterile reverse osmosis (RO) water and dried on sterile filter paper. Half of the seeds were lightly macerated in a sterile mortar and pestle, and were, together with the remaining whole seeds, spread evenly in separate sterile Petri dishes containing 1.3% (w/v) nutrient agar (NA) or 2.4% (w/v) potato dextrose agar (PDA). The second group of non-surface sterilized seeds was spread in a similar manner either lightly macerated or whole on NA or PDA.
(43) The Petri dishes were incubated in the dark at 25° C. (NA) or 20° C. (PDA) and checked regularly for approximately 4 wk. As soon as bacteria or fungi emerged from the seeds, they were sub-cultured individually onto sterile NA (bacteria) or PDA (fungi), and were incubated as described above to obtain pure cultures. For long-term storage of the bacteria, a single colony was grown overnight in sterile 2.5% (w/v) Luria-Bertani Miller Broth (LB) on a shaker at 180 rpm, 25° C. in dark. The culture was stored in sterile 25% (v/v) glycerol at −80° C.
(44) A total of 1485 microbes were isolated onto standard microbiological media and pure cultures were obtained. They consisted of: 1101 isolates of bacteria 384 isolates of fungi.
(45) Putative taxonomic identities were assigned (as described in Example 2) to 731 bacteria and 234 fungi based on comparisons of their 16S ribosomal RNA (16S rRNA, bacteria only) or internal transcribed spacer (ITS, fungi only) DNA sequences, with those in the EzTaxon and/or GenBank databases. Bacillus was the predominant bacterial genus recovered. Only 13 isolates belonged to the genus Erwinia.
(46) DSM 32302 was isolated from forage rape seed obtained from PGG Wrightson Seeds Ltd, New Zealand.
(47) DSM 32304 was isolated from forage rape seed obtained from PGG Wrightson Seeds Ltd, New Zealand.
(48) DSM 32305 was isolated from turnip seed obtained from PGG Wrightson Seeds Ltd, New Zealand.
(49) DSM 32303 was isolated from kohlrabi seed obtained from South Pacific Seeds Ltd, New Zealand.
Example 2: Molecular Genetic Identification
(50) Isolates of Erwinia were identified by partial DNA sequence analysis of the 16S rRNA region, and genes for the heat shock protein dnaJ (dnaJ), glyceraldehyde-3-phosphate dehydrogenase (gapDH) and recombinase A (recA). PCR amplifications were performed on a single colony grown overnight on NA at 25 or 28° C. in the dark. For the 16S rRNA region, a direct colony PCR was carried out in 25 μL reactions containing 1.25 U of AccuSure DNA polymerase (Bioline), 1×AccuBuffer (Bioline), 6.25 nmol of each dNTP (Bioline) and 5 pmol of primer pair f8-27 and r1510 (Invitrogen; Lipson and Schmidt 2004). These were incubated in a thermal cycler for 10 min at 95° C., followed by 30 cycles of 1 min at 95° C., 1 min at 55° C. and 2.5 min at 68° C., and then 10 min at 68° C.
(51) For the other genes, DNA extraction from the colony and subsequent PCR amplification of the DNA with 5 pmol of each primer (
(52) Amplification products were purified with Agencourt AMPure or Agencourt AMPure XP (Beckman Coulter) according to the manufacturer's instructions. Purified products were sequenced in the forward direction by Macrogen Inc (South Korea) or Lincoln University Sequencing Facility (New Zealand).
(53) E. persicina isolates ICMP 8932 and ICMP 12532, and Erwinia rhapontici isolate ICMP 15975 (Landcare Research) were also characterised. Genomic DNA was isolated from a culture grown overnight in LB on a shaker at 180 rpm, 25° C. in the dark with the Gentra Puregene Yeast/Bact. kit (Qiagen) following the manufacturer's instructions. PCR amplification of the DNA (10 ng) was carried out with the REDExtract-N-Amp Plant PCR kit as described above, only for the 16S rRNA region, reactions were incubated in a thermal cycler for 3 min at 94° C., followed by 35 cycles of 1 min at 94° C., 1 min at 55° C., and 2 min at 72° C., and then 10 min at 72° C.
(54) The DNA sequences from the Erwinia isolates were compared with the corresponding sequences from E. persicina (ICMP 8932 and ICMP 12532), E. rhapontici (ICMP 15975), and type strains of other Erwinia taxa and Xanthomonas campestris pv. campestris (Xcc; available from GenBank, National Center for Biotechnology Information, USA). These were aligned in Sequencher (Gene Codes Corporation) using the dirty data assembly algorithm, and assembly parameters of 60% minimum match and minimum overlap of 50. Some manual adjustments were made to the alignments to reposition or remove gaps.
(55) Phylogenetic trees were estimated from the alignments of each gene in MEGA6 (Tamura et al. 2013) using the Maximum Likelihood method based on the Tamura 3-parameter model (Tamura 1992). A discrete Gamma distribution with 5 rate categories was used to model evolutionary rate differences among sites. All positions containing gaps were eliminated. The initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood approach. The robustness of the tree was measured by the Bootstrap method with 1000 replications. A Bootstrap value of 70% or greater was considered well supported. Xcc type strain ICMP 13 was used as the outgroup for rooting the tree.
(56) Erwinia 75, 76, 90 and 1859 isolates displayed 100% sequence identity to the type strain of E. persicina (ICMP 12532). These isolates clustered in the phylogenetic trees with this type strain to form a well-supported group separate from most other Erwinia taxa (
(57) SEQ ID NO. 1 to 4 were used to characterise DSM 32302, SEQ ID NO. 5 to 8 were used to characterise DSM 32304; SEQ ID NO. 9 to 12 were used to characterise DSM 32305 and SEQ ID NO. 13 to 16 were used characterise DSM 32303.
(58) Alignments of the sequences of SEQ ID NO: 1 to 16 are shown in
Example 3: In Vitro Screening
(59) Bacterial isolates representative of the range of taxa present in brassicas were evaluated in dual culture assays against Xcc isolates Xcc2 (I. Harvey, PLANTwise), ICMP 2 and/or ICMP 4013 (Landcare Research), and against Sclerotinia sclerotiorum (Ss) isolates LU462 and LU471 from kale (Lincoln University Culture Collection).
(60) For each Xcc isolate, inoculum grown on yeast dextrose chalk agar (YDCA) at 25° C. in the dark for 3-5 d, was resuspended in 0.1 M MgSO4 and adjusted to an optical density of 0.80±0.01 at 600 nm (estimated concentration of 2×10.sup.8 CFU/mL). This inoculum (0.1 mL) was spread over the agar surface in separate sterile Petri dishes containing either YDCA or PDA. The test bacteria were introduced soon after.
(61) Bacterial cells grown on NA at 25° C. in the dark for 1-5 d were applied, using an inoculation loop, to the Xcc-inoculated Petri dishes at four equidistant inoculation points, 18 mm from the edge. For each bacterial isolate, two Petri dishes (2×YDCA, or in later experiments 1×YDCA and 1×PDA) were prepared against each Xcc isolate. The Petri dishes were incubated in a random order at 25° C. in the dark.
(62) In the dual culture assays with Ss, separate sterile Petri dishes containing PDA were inoculated with the bacterial isolates as described above, and were incubated overnight at 25° C. in the dark before the pathogen was introduced. A mycelial disc of Ss (6 mm in diameter) was removed from a culture grown on PDA at 20° C. in the dark for 4-6 d and transferred to the centre of the Petri dish with the test bacteria. Two Petri dishes were prepared for each bacterial isolate against each Ss isolate, and were incubated in a random order at 20° C. in the dark.
(63) The dual culture assays were assessed 2-8 d after pathogen inoculation. The bacterial isolates were given scores in the assays against Xcc as 0=no inhibitory effects on Xcc growth, 1=small effects, 2=moderate effects, or 3=large effects. Against Ss, they were scored as 0=no inhibitory effects on Ss growth, 1=Ss and test bacterium approach one another and stop growing, or 2=Ss growth is inhibited at a distance leaving a clear zone of inhibition or becomes overgrown by the test bacterium.
(64) The bioactivity scores of the bacterial isolates in each dual culture assay were statistically analysed using an analysis of variance (ANOVA) for a completely randomised experimental design with a treatment structure of 2 (pathogen isolate)×>1 (test isolate). For those dual culture assays carried out on two different media, the treatment structure was amended to 2 (media)+2 (pathogen isolate)×>1 (test isolate). Test isolates that exclusively scored zero, or conversely, the greatest bioactivity score, were omitted from ANOVA to avoid violating the assumption of equal variance. These were compared to the variable treatments using the least significant effect (LSEffect 5%), that is the least significant difference (LSD 5%) divided by the square root of 2.
(65) A total of 38 bacterial isolates showed bioactivity against both pathogens in vitro (
(66) Some of the bacterial isolates only displayed antagonism towards one pathogen, and these included, in addition to some of the aforementioned genera, isolates from the bacterial genera Chryseobacterium, Pantoea and Variovorax (
Example 4: Bioactivity in Seedling Bioassays with Xcc
(67) The bioactivity of E. persicina isolates 75, 76, 90 and 599 were, in addition to a number of other bacterial isolates, evaluated against Xcc isolates ICMP 4013 and ICMP 6497 (Landcare Research) in cabbage and forage rape seedling bioassays.
(68) Xcc inoculum was prepared from YDCA cultures that had been grown in the dark at 25° C. for 3 d. The inoculum, resuspended in sterile 0.1% (w/v) bacteriological peptone (BP) water, was adjusted to a concentration of 1×10.sup.7 colony forming units (CFU)/mL based on its optical density at 600 nm.
(69) Seeds from cabbage and forage rape were surface-sterilised in 1% NaOCl with 0.01% (v/v) Tween 20. Xcc inoculum (1×10.sup.7 CFU/mL) or sterile BP water (negative control) was applied to the surface-sterilized seed at a rate of 3 mL/g seed under vacuum at 6.7 kPa with continuous mixing for 5 min. The seeds were collected in sterile Miracloth and dried overnight in open Petri dishes in a laminar flow cabinet.
(70) The bacterial isolates were grown in 100 mL of LB on a shaker at 180 rpm, 30° C. in the dark for 18 h. The bacterial cells were collected from the culture by centrifugation at 3,220×g for 20 min, washed with sterile BP water, and centrifuged again before resuspending in sterile BP water. The inoculum was adjusted to a concentration of 1×10.sup.8 CFU/mL based on its optical density at 600 nm and applied to the Xcc-inoculated seeds at a rate of 0.6 mL/g seed. Sterile BP water was applied to the negative and positive controls. The seeds were mixed manually with the inoculum and incubated overnight in a closed but not sealed Petri dish in a laminar flow cabinet.
(71) For each seed treatment, 25 seeds were evenly spaced on two layers of germination blotter (60 mm×90 mm, Anchor Steel Blue Blotter, Anchor Paper Company) moistened with 10 mL of sterile RO water. The blotters and seed were transferred to a clean plastic container with clear sides and an additional 3 mL of sterile RO water was added before sealing the container.
(72) A minimum of 10 germination blotters were prepared for each seed treatment. Assays were arranged in a randomised complete block design at 30° C. light (1000 lux) for 8 h and 20° C. dark for 16 h. In order to minimize the variance of the difference between the control and treatment, the number of positive and negative controls in each block was approximately equal to the square root of the total number of treatments.
(73) Germination was assessed 5 d after sowing (DAS) according to the International Seed Testing Association (ISTA) guidelines (Don, 2009). The occurrence of disease symptoms was assessed in normal seedlings 8 DAS. Symptoms typically manifested as a transparent to light brown lesion on the upper hypocotyl.
(74) The percentage germination and disease incidence was statistically analysed using an ANOVA for a randomised complete block design with 10 blocks+>1 (test isolate). Treatments that consistently had germination or disease levels close to 0 or 100% were omitted from the analysis to avoid violating the assumption of equal variance. These were statistically compared to the variable treatments using the LSEffect 5%.
(75) Combined analysis of germination and disease incidence in different brassica species, against different pathogen isolates and overall, were carried out on the data means for each isolate in each assay using an unbalanced analysis of variance. In cases where multiple seed lots or pathogens were tested in the same assay, the main effect means for the isolates were used in order to achieve independence in the data. All statistical analyses were performed using GenStat.
(76) E. persicina isolates 75, 76 and 90 reduced the incidence of black rot in cabbage and/or forage rape seedlings on average by 88-99% (
(77) Seedling emergence was high from seed treated with E. persicina isolates 75, 76 and 90 (
Example 5: Biocontrol of Xcc in Cabbage
(78) E. persicina isolates 76 and 90 were evaluated, among other bacterial and fungal isolates, for biocontrol activity in cabbage against Xcc isolates ICMP 6497 and ICMP 21080 (Landcare Research).
(79) The pathogen was applied to cabbage seed together with E. persicina isolates 76 and 90 and other bacterial and fungal isolates following the methods described in Example 4 with some modifications. The inoculum of Xcc was increased to a concentration of 1×10.sup.9 CFU/mL and that of the bacterial and fungal isolates to 5×10.sup.8 and/or 5×10.sup.9 CFU/mL.
(80) The treated seed was sown in 2×2 cell trays containing 25 mL/cell of saturated potting mix (pH 5.8). Two seeds were sown in each cell to a depth of 10 mm and was thinned to one normal seedling per cell after 1 wk. Each cell tray was placed on an individual saucer. The potting mix was composed of Kiwipeat (600 L/m.sup.3, New Zealand Growing Media), pumice (400 L/m.sup.3, Egmont Commercial), Osmocote Exact Mini (1.5 kg/m.sup.3, Everris International), dolomite lime (5 kg/m.sup.3, Golden Bay Dolomite), finely ground agricultural lime (2 kg/m.sup.3, Oxford Lime Company), superphosphate (1 kg/m.sup.3, Ravensdown) and Hydraflo (1 kg/m.sup.3, Everris International).
(81) The pot trials were arranged following a randomised complete block design in a growth room (BDW120 Plant Growth Cabinets; Conviron) at the New Zealand Biotron (Lincoln University). Conditions in the growth room cycled from 25° C. light (400 μmol/m.sup.2/s) for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. In order to minimize the variance of the difference between the control and treatment, the number of positive controls (and sometimes negative controls) in each block was approximately equal to the square root of the total number of treatments.
(82) The pot trials were lightly watered overhead with a hand-held watering wand 1 DAS. Thereafter, they were watered as required to maintain the potting mix in a moist condition. Liquid fertiliser (Agrichem High NK, PGG Wrightson Turf) was used at weekly intervals from 2-3 wk after sowing. The fertiliser, diluted 1:200, was applied to the pot trials at sufficient levels to saturate the potting mix and was gradually increased over time to fill the saucer.
(83) Seedling emergence was assessed 7-8 DAS and were according to their above ground appearance, categorised as normal or abnormal following the International Seed Testing Association (ISTA) guidelines for Brassica seedlings (Don, 2009). Normal seedlings were assessed for black rot disease symptoms at weekly intervals from 14 DAS onwards. The presence of characteristic V-shaped chlorotic lesions and blackened veins (Rimmer et al. 2007) were recorded for up to 21 DAS on the cotyledons and 42 DAS on the true leaves.
(84) The percentage emergence and disease incidence was statistically analysed using an ANOVA as described in Example 4. Disease incidence was based on the cumulative total of infected plants across successive weeks.
(85) In warm, humid conditions that favour the disease, E. persicina isolates 76 and 90 significantly decreased black rot levels by 80-98% when applied at different rates (
(86) There were no negative effects on emergence with E. persicina isolate 76 (
Example 6: Effect of Application Rate on Symptom and Latent Xcc Infection
(87) The ability of E. persicina isolates 76, 90, 1774 and 1860 when applied to seed at different rates, to control both symptom and latent Xcc infections in cabbage were compared.
(88) The pot trial was conducted as described in Example 5 with some amendments. Cabbage seed was artificially inoculated with Xcc isolate ICMP 21080 (Landcare Research). E. persicina was applied to this seed at six different concentrations; 5×10.sup.4, 5×10.sup.5, 5×10.sup.6, 5×10.sup.7, 5×10.sup.8 and 5×10.sup.9 CFU/mL.
(89) The seedlings were assessed weekly for black rot symptoms in the cotyledons and true leaves until 28 and 42 DAS, respectively. The occurrence of latent Xcc infections were tested in seedlings treated with E. persicina at concentrations of ≥3×10.sup.6 CFU/g seed and in the controls. One seedling (or two positive control seedlings) that had not displayed disease symptoms throughout the pot trial was randomly selected from each block. The vascular fluid was extracted from the plant using a Scholander pressure chamber (Plant Water Status Console 3000F01, ICT International).
(90) The plant cut at the base of the stem just above the potting mix, was mounted inside the pressure chamber. The stem inserted in a short length of sterile silicon-rubber tubing, was threaded through the specimen holder into a sterile 1.7 mL collection tube. A total of 2,760 kPa was applied to the chamber for 2 min or longer if necessary, to collect >0.1 mL of vascular fluid. Appropriate 10-fold serial dilutions of the vascular fluid were spread (0.1 mL) over the agar surface of sterile Petri dishes containing FS agar medium. The occurrence of Xcc was determined after 3 d at 28° C. in the dark. The cultures were examined for small, pale, mucoid colonies surrounded by a zone of starch hydrolysis.
(91) The percentage emergence was statistically analysed using an ANOVA for a randomised complete block design with 15 blocks and a factorial treatment structure of 4 (E. persicina isolate)×6 (rate)+1 (positive control)+1 (negative control). The E. persicina isolates 76, 90, 1774 and 1860 were applied at six target rates of 3×10.sup.4, 3×10.sup.5, 3×10.sup.6, 3×10.sup.7, 3×10.sup.8 and 3×10.sup.9 CFU/g to seed artificially inoculated with Xcc isolate ICMP 21080. Also included were seed treated only with Xcc (positive control) or BP water (negative control). For the rate factor, linear and quadratic contrasts were included in the analysis, as well as contrasts to examine the effects of the E. persicina isolates. All statistical analyses were performed using GenStat.
(92) The negative control was omitted from the ANOVA of the percentage of symptom and latent infections, and total disease incidence. This was necessary due to the absence of infection, to avoid violation of the ANOVA assumption of equal variance. This treatment was statistically compared to the variable treatments using LSEffect 5%. The percentage of symptom infections was based on the cumulative total of plants with symptoms across successive weeks. The total disease incidence was calculated based on the total number of plants with symptoms and latent infections. The latter was estimated for each treatment in each block by multiplying the number of symptomless plants by the proportion of plants with latent infections. The rate factor in the factorial treatment structure was reduced to four for ANOVA of the percentage latent infection and total disease incidence.
(93) The biocontrol activity of E. persicina isolates 76 and 90 against Xcc differed significantly from E. persicina isolates 1774 and 1860 (p<0.001,
Example 7: Impact of Temperature on Biocontrol Activity
(94) The efficacy of E. persicina isolate 76 and other BCAs when applied at different rates to Xcc-inoculated cabbage seed were compared under two different temperature regimes.
(95) The pot trial was conducted as described in Example 5 with some amendments. Cabbage seed was artificially inoculated with Xcc isolate ICMP 6497 (Landcare Research). E. persicina isolate 76 and three other BCAs were applied to the seed at concentrations of 5×10.sup.7, 5×10.sup.8 and 5×10.sup.9 CFU/mL. One of the pot trials was held in a growth room under the same conditions as described in Example 5. For the other pot trial, growth room conditions cycled from 20° C. light (400 μmol/m.sup.2/s) for 13 h to 10° C. dark for 11 h.
(96) The percentage emergence at the two temperature regimes was analysed together using an ANOVA for a randomised complete block design with 2 (main plots)+10 (blocks) and a factorial treatment structure of 2 (temperature regime)×(4 (BCA isolate)×3 (low, medium and high rate)+1 (Xcc inoculant)+1 (BP inoculant)). The main plots were the 2 temperature regimes of 20° C. D/10° C. N and 25° C. D/15° C. N. The four BCA isolates, including E. persicina isolate 76, were applied at three target rates; low: 3×10.sup.7 CFU/g; medium: 3×10.sup.8 CFU/g; and high: 3×10.sup.9 CFU/g. Also included were seeds treated with inoculants Xcc isolate ICMP 6497 or BP water. For the rate factor, linear and quadratic contrasts were included in the analysis, as well as contrasts to examine the effects of BCA and Xcc inoculant. All statistical analyses were performed using GenStat.
(97) For ANOVA of the percentage disease incidence which was based on the cumulative total of infected plants across successive weeks, 13 treatments that were derived from seed pre-treated with Xcc inoculant were included in the analysis. There were no symptoms detected in the negative control (BP inoculant) and to avoid violation of the ANOVA assumption of equal variance, this treatment was omitted from the analysis. ANOVA was performed as described for emergence using a 2 (temperature regime)×(4 (BCA isolate)×3 (high, medium and low rate)+1 (Xcc inoculant)) factorial treatment structure.
(98) Application of E. persicina isolate 76 to seed reduced black rot in cabbage seedlings (
(99) The presence of E. persicina isolate 76 did not affect emergence of cabbage seed under warmer or cooler temperature regimes (
Example 8: Impact of pH on Biocontrol Activity
(100) The effect of pH on the biocontrol activity of E. persicina isolate 76 against black rot in cabbage was investigated together with another BCA.
(101) The pot trial was conducted as described in Example 5 with some amendments. Cabbage seed was artificially inoculated with Xcc isolate ICMP 6497 (Landcare Research) and treated with E. persicina isolate 76 and one other BCA. These were sown in potting mix of pH 5.0, pH 5.8 and pH 6.4. The potting mix pH was reduced to pH 5.0 by excluding the agricultural lime and decreasing the levels of dolomite lime to 3 kg/m.sup.3, and was raised to pH 6.4 by increasing the levels of both agricultural lime and superphosphate to 7 kg/m.sup.3. The potting mix pH was tested at the start and end of the pot trials following the Australian Standard for Potting Mixes (AS 3743-2003).
(102) The percentage emergence in the pH pot trials was statistically analysed using an ANOVA for a randomised complete block design with 15 blocks and a 3 (pH)×4 (2 (BCA isolate)+1 (Xcc inoculant)+1 (BP inoculant)) factorial treatment structure. The pH of the potting mixes were pH 5.0, 5.8 or 6.4. The BCA isolates were E. persicina isolate 76 and one other BCA. Also included were seeds treated with inoculants Xcc isolate ICMP 6497 or BP water. Linear and quadratic polynomial contrasts of the pH factor, and contrasts to examine the effects of BCA, BCA isolate and Xcc inoculant were included in the analysis.
(103) For ANOVA of the percentage disease incidence which was based on the cumulative total of infected plants across successive weeks, 9 treatments that were derived from seed pre-treated with Xcc inoculant were included in the analysis. There were no symptoms detected in the treatment with BP water inoculant at the different pH levels and to avoid violation of the ANOVA assumption of equal variance, this treatment was omitted from the analysis. ANOVA was performed as described for emergence using a 3 (pH)×3 (2 (BCA isolate)+1 (Xcc inoculant)) factorial treatment structure.
(104) The potting mixes were at the start and end of the pot trial close to the target pH levels of 5.0, 5.8 and 6.4. In the absence of BCA, the level of black rot in cabbage was significantly higher at pH 6.4 than pH 5.0 and 5.8 (p=0.004,
(105) E. persicina isolate 76 resulted in a 93-100% reduction in disease levels across all pH levels (
(106) The rate of emergence of cabbage was high across all pH levels in the presence of E. persicina isolate 76 (
Example 9: Biocontrol Activity Under Wet Conditions
(107) The biocontrol activity of 13 isolates of E. persicina from brassica (75, 76, 90, 152, 235, 376, 599, 1601, 1657, 1774, 1859, 1860 and 1953) was evaluated against Xcc isolate ICMP 21080 (Landcare Research).
(108) The pot trials were carried out as described in Example 5, with some exceptions. The seeds were inadvertently covered after Xcc inoculation. The pot trial was carried out in 3×6 cell trays and only a single seed was sown in each cell. The potting mix was kept excessively wet during the course of the pot trial. The true leaves of seedlings were only assessed for black rot symptoms up to 30 DAS.
(109) The percentage emergence and disease incidence were statistically analysed using an ANOVA for a randomised complete block design with five blocks and 15 treatments. The treatments included the positive and negative controls, and E. persicina isolates 75, 76, 90, 152, 235, 376, 599, 1601, 1657, 1774, 1859, 1860 and 1953.
(110) The seedlings were overwatered and disease levels 30 days after sowing (DAS) were high, reaching from 95% in the positive control (
(111) Under these conditions, four of the Erwinia isolates; 75, 76, 90 and 1859, significantly reduced symptom infections by Xcc isolate ICMP 21080 (
(112) There were no negative effects on emergence with the different Erwinia isolates (
Example 10: Effect of Application Method on Symptom and Latent Xcc Infection
(113) The efficacy of E. persicina isolate 76 when applied to the seed and/or sowing hole against both symptom and latent Xcc infection was investigated under greenhouse and growth room conditions, together with two other BCAs
(114) Cabbage seed was inoculated with Xcc isolate ICMP 21080 (Landcare Research) and treated with BP water, E. persicina isolate 76, or one of two other BCAs as described in Example 5. For potting mix application, inoculum was prepared in the same way to a target concentration of 2×10.sup.7 CFU/mL and applied to the potting mix 1 DAS. The 2×2 cell trays were filled with saturated potting mix (pH 5.8, see Example 5) and a total of 2×10.sup.7 CFU were applied to the sowing hole of each 25 mL cell. The cells trays were stored in plastic bags at ambient until the seed was sown the next day as described in Example 5.
(115) The seedlings were raised as described in Example 5, only one of the pot trials was held in a Durolite-clad greenhouse at Lincoln University (New Zealand). The set point temperatures for heating and venting of the greenhouse were 17 and 24° C., respectively.
(116) Seedling emergence and the occurrence of black rot disease symptoms were assessed as described in Example 5. There were some exceptions. In the growth room, disease symptoms in the true leaves were assessed up to 40 DAS. Emergence was assessed 9 DAS in the greenhouse, and disease symptoms in the cotyledons and true leaves up to 35 and 49 DAS, respectively.
(117) Seedlings were tested for the presence of latent infections. One seedling that had not displayed disease symptoms throughout the pot trial was randomly selected from each cell tray. In addition, a random selection of diseased seedlings was tested as positive controls. Seedlings were sampled 41-46 DAS from the pot trial in the growth room and 50-65 DAS from the pot trial in the greenhouse. Fluid was extracted from the vascular vessels of the plant shoots following the methods described in Example 6.
(118) The percentage emergence was statistically analysed using an ANOVA for a randomised complete block design with 15 blocks in the growth room and 40 blocks in the greenhouse, and a 3 (BCA isolate)×3 (application method)+1 (Xcc inoculant)+1 (BP inoculant) factorial treatment structure. The BCA isolates were E. persicina isolate 76 and two other BCAs. Also included were seeds treated with inoculants Xcc isolate ICMP 21080 or BP water. Contrasts to examine the effect of seed or potting mix applications in the application method factor, and of BCA, BCA isolate and Xcc inoculant were included in the analysis.
(119) For ANOVA of the percentage symptom and latent infections and total disease incidence in the growth room, and percentage symptom infections in the greenhouse, the BP inoculant factor was omitted from the factorial treatment structure. This was necessary due to the absence of infection, to avoid violation of the ANOVA assumption of equal variance. This treatment was statistically compared to the variable treatments using the LSEffect 5%. ANOVA of the percentage latent infections and total disease incidence in the greenhouse was performed as described for emergence. The percentage of symptom infections was based on the cumulative total of infected plants across successive weeks. The total disease incidence was calculated based on the total number of plants with symptom and latent infections. The latter was estimated for each treatment in each block by multiplying the number of symptomless plants by the proportion of plants with latent infections.
(120) The method of application significantly affected emergence of cabbage seed in the greenhouse but not in the growth room (
(121) In both the greenhouse and growth room, E. persicina isolate 76 had a major effect on disease incidence, causing a decrease in both symptom and latent Xcc infections (
Example 11: Compatibility with Agrichemicals
(122) The efficacy of E. persicina isolate 76 against Xcc isolate ICMP 21080 (Landcare Research) was assessed in the greenhouse under a chemical spray programme used in a commercial nursery for raising brassica transplants.
(123) E. persicina isolate 76 was applied to cabbage seed artificially inoculated with Xcc isolate ICMP 21080 following the methods described in Example 5, only the seeds were held at ambient temperature for 1 d and then at 4° C. for 4 d before they were sown. A single seed was sown in each cell of a 2×2 cell tray and 10 cell trays of the same treatment were placed together on a plastic tray. The trays were arranged in a Durolite-clad greenhouse at Lincoln University (New Zealand) following a randomised complete block design with a total of 8 blocks. In each block the unsprayed treatments were replicated twice to minimize the variance of the difference between these and the sprayed treatments. The set point temperatures for heating and venting of the greenhouse were 17 and 24° C., respectively.
(124) The pot trial was watered and fertilised as described in Example 5 with at least one watering between fertiliser and chemical spray applications. Care was taken to ensure the seedlings were not water stressed at the time of spraying and that the foliage was dry. Chemical sprays were applied weekly to the selected seedlings starting 9 and 16 days after sowing as outlined in
(125) The seedlings were assessed as described in Example 5.
(126) The percentage emergence was statistically analysed using an ANOVA for a randomised complete block design with eight blocks and two treatments. The treatments were Xcc-inoculated seed treated with or without E. persicina isolate 76. For ANOVA of the percentage disease incidence, the factorial treatment structure of 2 (seed inoculant)×3 (spray) was used. Seedlings from Xcc-inoculated seed treated with or without E. persicina isolate 76 were left unsprayed or sprayed weekly with chemicals starting 9 or 16 DAS. For the spray factor, contrasts were included to examine the effects of spraying and spray timing.
(127) The chemical spray programme had no effect on the efficacy of E. persicina isolate 76 (
Example 12: Plant Growth Promotion
(128) E. persicina isolates 75, 76, 90 and 599, together with some other bacterial isolates, were evaluated for their ability to promote cabbage plant growth in the greenhouse.
(129) Cabbage seeds were surface-sterilized and inoculated with the bacterial isolates following the methods described in Example 4. The treated seeds were sown in moist potting mix in 0.9 L plastic planter bags (Egmont Commercial). Six seeds were sown in each bag to a depth of 10 mm and were thinned to one randomly selected normal seedling 8 DAS. The potting mix was composed of Kiwipeat (600 L/m.sup.3, New Zealand Growing Media), pumice (400 L/m.sup.3, Egmont Commercial), Osmocote Exact Mini (1.5 kg/m.sup.3, Everris International), dolomite lime (4 kg/m.sup.3, Golden Bay Dolomite), and Hydraflo (1 kg/m.sup.3, Everris International). Each bag was placed on a saucer and water was applied overhead as required to maintain the potting mix in a moist condition.
(130) The pot trial was conducted in a Durolite-clad greenhouse at Lincoln University (New Zealand). The set point temperatures for heating and venting of the greenhouse were 17 and 24° C., respectively. The pot trial was split into two experiments according to harvest date (22 or 43 DAS). Each experiment was arranged in a randomised complete block design with 10 blocks. In order to minimize the variance of the difference between the negative controls and treatments, there were three negative controls in each block.
(131) Seedling emergence was assessed 7 DAS as described in Example 5. The pot trials were harvested at 22 and 43 DAS. The number of completely unfurled leaves on the plant was recorded. The dry weights of the roots and shoots were measured after complete drying at 65-70° C. The roots were carefully washed in water to remove the potting mix before drying.
(132) The percentage seedling emergence, number of leaves and shoot and root dry weights were statistically analysed using an ANOVA for randomised complete block design with a treatment structure of 10 (replicate)+5 (bacterial isolate). A combined analysis of emergence was carried out on the data means for each isolate for the two harvest dates.
(133) There were no negative effects observed on cabbage emergence and growth with E. persicina (
Example 13: Seed Coating Formulation
(134) The efficacy of a seed coating formulation of E. persicina isolate 76 against Xcc isolate ICMP 21080 (Landcare Research) were compared with the seed treatment described in Example 5. A second BCA was also tested.
(135) For formulation as a seed coating, cells of E. persicina isolate 76 and the other BCA were formulated as described for Formulation 5 in Swaminathan et al. (2015). This formulation was applied to untreated (bare) cabbage seed and seed artificially inoculated with Xcc isolate ICMP 21080 following the methods described in Example 5.
(136) E. persicina isolate 76 and the other BCA were also applied to the seed with or without Xcc following the standard seed treatment method described in Example 5, only three different concentrations of the BCA were used; 5×10.sup.7, 5×10.sup.8 and 5×10.sup.9 CFU/mL.
(137) The pot trials were conducted and assessed as described in Example 5.
(138) The percentage emergence was statistically analysed using an ANOVA for a randomised complete block design with 15 blocks and a 2 (formulation)×2 (Xcc presence or absence)×(2 (BCA isolate)×3 (low, medium and high rate)+1 (BCA absence)) factorial treatment structure. Formulations were the seed coating and standard seed treatment and were applied to seed inoculated with Xcc isolate ICMP 21080 and dried overnight, or to bare seed. The BCA, E. persicina isolate 76 and one other BCA, were applied at three target rates; low: 3×10.sup.7 CFU/g; medium: 3×10.sup.8 CFU/g; and high: 3×10.sup.9 CFU/g. Also included was seed not treated with BCA. For the rate factor, linear and quadratic polynomial contrasts were included in the analysis.
(139) For ANOVA of the percentage disease incidence, only 14 treatments that were derived from seed pre-treated with Xcc inoculant were included in the analysis. The remaining 14 treatments that were derived from bare seed, were omitted to avoid violation of the ANOVA assumption of equal variance. No symptoms were detected in 12 of the omitted treatments and in the remaining 2 treatments, symptoms occurred in 3% of plants. ANOVA was performed as described for emergence using the same contrasts and a 2 (formulation)×(2 (BCA isolate)×3 (high, medium and low rate)+1 (BCA absence)) factorial treatment structure.
(140) The seed coating formulation of E. persicina isolate 76 displayed high levels of disease control comparable to that of the standard seed treatment (
(141) Neither BCA or application rate had a major effect on emergence but emergence was affected by formulation (
Example 14: Formulation and Application of E. persicina
(142) The efficacies of granule and freeze-dried formulations of E. persicina isolate 76 against Xcc isolate ICMP 21080 (Landcare Research) were compared to the standard non-formulated preparation. The individual and combined effects of applying formulated and non-formulated inoculum to the seed and potting mix, and as a drench and foliar spray were examined in a factorial design.
(143) For the granule formulation, cells of E. persicina isolate 76 were coated onto zeolite as described in patent WO2008023999 (Swaminathan and Jackson, 2008). For the freeze-dried formulation, cells of E. persicina 76 were freeze-dried in 5% (w/v) sucrose solution as described in Wessman et al. (2013). Suspensions of the freeze-dried formulation were prepared on the day of application in tap water at the target concentrations listed in
(144) The non-formulated inoculum was prepared following the methods described in Example 5 with some modifications. E. persicina isolate 76 was cultured in 500 mL of LB broth on a shaker at 250 rpm, 30° C. in the dark for 16 h. The inoculum was resuspended in sterile BP water adjusted to the target concentrations listed in
(145) Cabbage seeds were artificially inoculated with Xcc isolate ICMP 21080 and treated with suspensions of the freeze-dried and non-formulated inoculum of E. persicina isolate 76 following the methods described in Example 5. Seeds for their respective controls were treated with 0.7% (w/v) sucrose or BP water.
(146) The granule formulation and suspensions of the freeze-dried and non-formulated inoculum were incorporated by hand into the bulk and cover potting mix at the rates outlined in
(147) After sowing, suspensions of the freeze-dried and non-formulated inoculum were applied individually to the mix as a drench using a piston-pressurised hand sprayer (Solo 456, Solo NZ) and 22 d later to the seedlings as a foliar spray using a trigger pump sprayer (Jet500, McGregor). The rates used are as outlined in
(148) A factorial design of 2 (seed inoculant)×2 (seed formulation)×4 (bulk mix)×4 (cover mix)×3 (drench)×3 (foliar spray) was followed to prepare a total of 576 unique treatment combinations. Two treated seeds were sown in each cell to a depth of 10 mm in a 2×2 cell tray containing 25 mL of potting mix per cell. An additional 64 cell trays were prepared with seed from the negative control, half of which were treated with sucrose and the remaining with BP water. These were sown in moist untreated potting mix.
(149) After the drench was applied, the cell trays were placed inside plastic bags in a growth room overnight. The pot trial was, due to space constrants, distributed across two growth rooms (BDW120 Plant Growth Cabinets, Conviron) in the New Zealand Biotron (Lincoln University). Conditions in the growth rooms cycled from 25° C. light (400 μmol/m.sup.2/s) for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The entire pot trial was repeated in the nursery. The cell trays were initially placed in a Durolite-clad greenhouse but 5 DAS were moved to a glasshouse due to low light conditions. They were returned to the greenhouse for the final week of the pot trial. The cell trays were arranged in a completely randomised order on individual saucers. The negative control was randomly distributed among the other cell trays and used as an indicator of secondary spread.
(150) The pot trial was watered and fertilised as described in Example 5 and was thinned 7 DAS to one normal seedling per cell. The temperature and relative humidity were recorded every 30 min in the growth rooms and at the nursery with a datalogger (Hobo U23 Pro V2, Onset).
(151) Seedling emergence and the occurrence of black rot disease symptoms were assessed in the pot trials using methods similar to those described in Example 5. Disease assessments were carried out 15, 21 and 42 DAS.
(152) The percentage emergence was statistically analysed using an ANOVA for a complete randomised design with a factorial treatment structure of 2 (seed inoculant)×2 (seed formulation)×4 (bulk mix)×4 (cover mix)×3 (drench). A fifth factor of 3 (foliar spray) was added to the factorial treatment structure for ANOVA of the percentage disease incidence. The Xcc-inoculated seed was treated with or without E. persicina isolate 76 as a freeze-dried formulation or non-formulated preparation that contained sucrose or BP, respectively. The bulk and cover mixes were treated with water or E. persicina isolate 76 as a granule or freeze-dried formulation, or as a non-formulated preparation. The latter two treatments and water were applied as a drench and foliar spray. The two locations, growth room and greenhouse, were analysed separately, and for the former, the two growth rooms were used as a covariate for ANOVA. Contrasts were included in the analysis of the bulk mix, cover mix, drench and foliar spray factors to examine the effects of E. persicina and formulation. The percentage of disease incidence was based on the cumulative total of seedlings with symptoms across successive weeks. All statistical analyses were performed using GenStat.
(153) The average temperature and relative humidity of the growth rooms were higher than at the nursery.
(154) Emergence was high for the different formulations and methods of application of E. persicina isolate 76 both in the growth room and glasshouse (
(155) Both in the growth room and glasshouse, application of E. persicina to seed was the main factor affecting disease incidence (
(156) In the absence of a seed application, the addition of E. persicina isolate 76 as a freeze-dried formulation or a non-formulated preparation to the bulk mix in the growth room and cover mix in the glasshouse, significantly reduced disease levels compared to the positive control (
(157) Addition of the granule formulation of E. persicina to the bulk and cover mixes in the glasshouse and to the bulk mix in the growth room, significantly increased disease levels compared to the freeze-dried formulation and non-formulated preparation (
(158) There was no evidence that application of E. persicina as a drench after sowing or as a foliar spray 22 DAS reduced the incidence of disease (
Example 15: Biocontrol Activity in Nursery-Raised Seedling Transplants
(159) The ability of E. persicina isolate 76 to prevent symptomless spread of Xcc in cabbage seedlings during transplant-raising in the nursery was investigated in two pot trials conducted under different watering regimes.
(160) For both pot trials, E. persicina isolate 76 was applied as a seed treatment to cabbage seed naturally infected with Xcc. Inoculum of E. persicina isolate 76 was prepared at a concentration of 5×10.sup.9 CFU/mL in non-sterile tap water using freeze-dried cells of this isolate. In the first pot trial, the commercial sticker Peridiam (6.67 mg/mL, Bayer) and Red dye (6.67 mg/mL, Bayer) were added to half of the inoculum. The inoculum was applied to the seed at a rate of 0.6 mL/g seed and dried overnight in a closed but not sealed Petri dish in a laminar flow cabinet. In the first pot trial, seed for the positive control was treated in a similar manner but without the BCA, whereas bare ‘untreated’ seed was used as the positive control in the second pot trial.
(161) The different seed treatments in the first pot trial were sown following different methods. For Method A, seed treatments with the sticker and dye were sown in 144 cell trays (25 mL per cell) containing potting mix used in a commercial nursery for brassica transplant raising. This potting mix was composed of peat (0.75 m.sup.3/m.sup.3, New Zealand Growing Media), blinding sand (particle size 1-4 mm, 0.2 m.sup.3/m.sup.3, North End Sand and Single Supplies), Yara PG Mix 12-14-24 (Orange, 1.2 kg/m.sup.3, Yara), Nutricote Micro TE 70 Day (1 kg/m.sup.3, Yates), dolomite lime (6.6 kg/m.sup.3, Ravensdown), gypsum (1.5 kg/m.sup.3, Ravensdown), rock phosphate (0.3 kg/m.sup.3, Summit-Quinphos) and Penetraide Re-Wetting Granules (0.5 kg/m.sup.3, Searles), and had a moisture content of 15%. For Method B, seed treatments without the sticker and dye were sown in 144 cell trays containing saturated in-house potting mix as described in Example 5. A single seed was sown in each cell to a depth of 10 mm and 14 cell trays were prepared for each of the four treatments in a replicate.
(162) The cell trays were placed in an unheated greenhouse with wind-break cloth ends and those sown in commercial potting mix (Method A) were watered within 20 min of sowing. After 2 wk in the greenhouse, the cell trays were moved to a shade house and grown for a further 4 wk. The trial was arranged in a split plot design with the positive control and BCA seed treatment forming the main plots, and Methods A and B the subplots. Plastic barriers were erected between the main plots to reduce the likelihood of cross-contamination. There were a total of three replicates. The set up of each replicate was staggered at 2 wk intervals with 4 wk between the sowing of the first and third replicate.
(163) In the second pot trial, bare ‘untreated’ seed and seed treated with E. persicina isolate 76 were sown in 144 cell trays containing commercial potting mix and watered within 20 min of sowing. For each replicate, two cell trays were prepared of each treatment. The trial was arranged in a split plot design with four replicates. One tray of each treatment in a replicate was placed in a growth room at the New Zealand Biotron (Lincoln University). Conditions in the growth room cycled from 25° C. light (400 μmol/m.sup.2/s) for 13 h to 15° C. dark for 11 h, with a constant relative humidity of 79%. The remaining trays were grown outside at the nursery at Lincoln University. The trays were placed in individual enclosures with half of the sides covered in plastic to prevent cross contamination between treatments and the remaining sides and top with vent netting to protect from cabbage white butterfly. Sticky yellow and blue insect traps (Egmont Commercial) were suspended in each enclosure to trap aphids, whitefly and thrips. The set up of the four replicates was staggered at 1 wk intervals. The seedlings were grown for 6 wk.
(164) The trials were watered as required to maintain the potting mix in a moist condition. In the first pot trial this was done manually overhead with a hand-held watering wand until the seedlings were moved to the shade house, where automated overhead micro-jet sprinklers were largely used. The second pot trial was watered over the surface of the potting mix until the seedlings emerged, after that it was watered from below. This involved manually filling the cell tray bases with water and then when the surface of the potting mix became moist, draining them of the excess water.
(165) Liquid fertiliser (diluted 1:200, Agrichem High NK, PGG Wrightson Turf) was applied overhead in first pot trial and from below in the cell tray bases in the second pot trial at weekly intervals starting 14-21 DAS. The chemical spray programme of a commercial nursery as described in Example 11 was followed in the first pot trial to control downy mildew and insect pests. The seedlings were sprayed weekly starting 14 DAS.
(166) For each of the trials, the temperature and relative humidity were recorded every 30 min using a datalogger (Hobo U23 Pro V2, Onset). In the second pot trial, the occurrence of surface moisture and guttation on the plants, and rainfall was recorded daily before 8 am.
(167) Seedling emergence was assessed 7-8 DAS as described in Example 4. The trials were assessed at different stages for black rot symptoms. The presence of characteristic V-shaped chlorotic lesions and blackened veins (Rimmer et al., 2007) were recorded once in the cotyledons and 2-3 times in the true leaves 20-23 and 20-44 DAS, respectively, in the first pot trial. Disease assessments were carried out on the true leaves at the end of the second pot trial (42 DAS).
(168) A random selection of seedlings that had not displayed symptoms were tested for the presence of Xcc and Erwinia species in the vascular fluid 43-51 DAS in the first pot trial and 42-46 DAS in second pot trial. Some seedlings with symptoms in the true leaves were also tested. Fluid was extracted from the vascular vessels of the plant shoots following the methods described in Example 6.
(169) The fluid was tested for Xcc by PCR amplification with the primer pairs Zup2311 and Zup2312 (Rijlaarsdam et al., 2004). DNA was extracted from the fluid (50 μL) and amplified with 0.25 μM of each primer using the REDExtract-N-Amp Plant PCR kit (Sigma-Aldrich) following the manufacturer's instructions. Reactions were incubated in a thermal cycler for 3 min at 94° C., followed by 35 cycles of 30 s at 94° C., 30 s at 60° C. and 1 min at 72° C., and then 10 min at 72° C.
(170) Amplification products (10 μL) were separated by agarose gel (1.5% w/v) electrophoresis in 1×TAE buffer, stained with ethidium bromide and visualized by UV transillumination on a VersaDoc Imager (Bio-Rad Laboratories). The molecular weight maker HyperLadder 50 bp (Bioline) was included on each gel for size determination of the products.
(171) The presence of Erwinia species in the vascular fluid was evaluated by PCR amplification with the primer pair Erwinia 1F (5′-AACCTTCGCTCAGTTTCCAG-3′) and Erwinia 1R (5′-CCTGACGTTCATCCACCAG-3′), designed to a protein of unknown function in E. persicina isolate 76. Reactions were conducted as described above for the Zup primer pair, except that the annealing temperature was raised to 63° C. The product, 263 bp in length, was detected by agarose gel electrophoresis.
(172) Standards of Xcc isolate ICMP 21080 (Landcare Research) and E. persicina isolate 76 were included in each PCR run. The inoculum used for these standards was prepared as described in Example 4, only in the second pot trial, the latter standards were prepared from the same inoculum used for the seed treatment. The inoculum was serially diluted 10-fold to obtain standards with concentrations ranging from 10 to 1×10.sup.6 CFU/mL.
(173) In first pot trial, the percentage emergence and incidence of Xcc and E. persicina isolate 76 was statistically analysed using an ANOVA for a split plot design with 3 (replicate)+2 (main plot)+2 (subplot) and a factorial treatment structure of 2 (seed treatment)×2 (method). The main plots were the seed treatment, either control or E. persicina isolate 76, and the subplots the method used to treat and grow the seed. In Method A, the seed treatment was applied in combination with a sticker and dye and grown in commercial potting mix, whereas in Method B, the seed treatment was applied in tap water alone and grown in saturated in-house potting mix. All statistical analyses involving ANOVA were performed using GenStat (VSN International).
(174) The incidence of Xcc in the first pot trial was divided into the percentage symptom infection, latent infection and total disease incidence. The total disease incidence was calculated based on the total number of plants with symptoms and latent infections. The latter was estimated for each treatment in each replicate by multiplying the number of symptomless plants by the proportion of plants with latent infections. A Chi-squared test was conducted to test the hypothesis that latent Xcc infection was related to whether or not Ep76 occurred in the vascular fluid of seedlings treated with this isolate using Method A.
(175) In the second pot trial, the percentage emergence and incidence of Xcc and E. persicina isolate 76, and frequency of leaf surface moisture and guttation, was statistically analysed using an ANOVA for a split plot design with 4 (replicate)+2 (main plot)+2 (subplot) and a factorial treatment structure of 2 (location)×2 (seed treatment). The main plots were the location, either the nursery or growth room, and the subplots the seed treatment, either control or E. persicina isolate 76.
(176) Emergence was high in both pot trials for seed treated with E. persicina isolate 76 (
(177) Disease symptoms were detected in <6% of seedlings in the first pot trial (
(178) Erwinia species were detected in the vascular fluid of 6 week old seedlings (
(179) In the second pot trial, the level of Xcc infection in cabbage seedlings after 6 weeks was low (
(180) Erwinia species occurred in <14% of seedlings in the second pot trial (
Example 16: Biocontrol Activity in the Field
(181) The ability of E. persicina isolate 76 to protect against natural seed-borne inoculum of Xcc and its impact on disease development in the field was investigated and compared to a second BCA.
(182) Two field trials were conducted at two different sites at Lincoln University (New Zealand). Cabbage seed naturally infested with Xcc was treated with E. persicina isolate 76 or another BCA following the methods described in Example 5. Following commercial practices, seedling transplants were raised in the nursery. The treated seed was sown in 144 cell trays containing 25 mL/cell of saturated potting mix (pH 5.8, see Example 5). A single seed was sown in each cell to a depth of 10 mm. The cell trays, arranged following a randomised complete block design, were initially placed in a Durolite-clad greenhouse, before being moved to an unheated greenhouse with wind-break cloth ends and/or a shade house, and then outside to be hardened. The seedlings were watered and fertilised as described in Example 5.
(183) In addition to the seed treatment, the BCAs were also applied to the foliage of seedling transplants raised for the second field trial. E. persicina isolate 76 was cultured in 250 mL of LB broth on a shaker at 200 rpm, 30° C. in the dark for 16 h. The concentration of bacterial inoculum was determined by measuring optical density of the culture at 600 nm. Based on this measurement, an appropriate volume of culture was combined with tap water and the sticker/wetting agent Bind-R-Duo (0.8 mL/L, SST New Zealand) to prepare a spray of 1×10.sup.11 CFU/L. The BCAs were only applied to foliage of seedlings grown from seed treated with the same isolate. The foliage was sprayed to run-off using a piston-pressurised hand sprayer (Solo 456, Solo NZ) with a water rate of 6.5 mL/s.
(184) The seedlings were mechanically transplanted in the field. For the first field trial, the first replicate was transplanted 42 d after sowing (DAS) and the remaining three replicates were, due to inclement weather conditions, transplanted 3 d later. The second field trial was transplanted 41 DAS. Only those seedlings that were likely to survive transplantation were transferred to the field. The field trials were arranged in a randomised complete block design with four blocks and around 600 plants per treatment per block.
(185) Prior to transplantation, fertilizers were applied to the soil to meet the nutrient requirements of cabbage. Herbicides were applied before and after transplantation for weed control. Once in the field, plants were irrigated using overhead sprinklers to maintain normal plant growth. Insecticides were applied as required both in the nursery and field to protect the plants from insect pests.
(186) The field trials were regularly assessed for the occurrence of black rot symptoms. In the second field trial assessments were only conducted after field transplantation.
(187) The percentage emergence and disease incidence was statistically analysed using an ANOVA for a randomised complete block design. Disease incidence was based on the cumulative total of infected plants across successive weeks. The first and last rows of plants in a plot were considered buffer plants and were excluded from the analysis. The average disease incidence was determined by calculating the area under the curve following the trapezoid rule and dividing by the number of days between the first and last assessment.
(188) Seed application of E. persicina isolate 76 with or without foliar applications during transplant raising, delayed the progression of black rot in the field (
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