the central North Island of New Zealand
J.L. Vanneste{ XE "Vanneste, J.L." }A, D.A. CornishA, J. YuA, and C.E. MorrisBA
The New Zealand Institute for Plant and Food Research Limited, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand
B
INRA, UR 407 Pathologie Végétale, F‐84140 Montfavet, France
INTRODUCTION
Epiphytotics of plant bacterial diseases can occur where
previously no or little inoculum was thought to be present. The
source of the primary inoculum of these epiphytotics is not
always easy to determine, especially for pathogenic bacteria,
which are good epiphytes, such as Pseudomonas syringae, one
of the most economically important bacterial plant pathogens.
This project aimed to determine whether rivers from the Central
North Island of New Zealand could constitute a reservoir for P. syringae. P. syringae is a complex group of strains which can be
grouped in about 50 different pathovars (strains which have the
same pathogenicity and the same host range) or in nine
genomospecies (strains which belong to the same species based
on DNA/DNA homology) (1). In this study we isolated some
strains of P. syringae and tried to determine to which
genomospecies or which pathovar they belong based on
polymerase chain reaction (PCR) experiments.
METHODS AND RESULTS
Collection of water samples and isolation of bacteria. Water
samples were collected from the Waikato River (Hamilton) and
from the Whakapapanui Stream (Tongariro National Park). The
isolation of bacteria was carried out as described previously (4).
Ten strains from Whakapapanui and five strains from the
Waikato River, which showed all the characteristics of strains of P. syringae: ability to produce a fluorescent pigment on a
modified King’s B medium, ability to cause a hypersensitive
reaction when infiltrated into tobacco plant, absence of a
cytochrome c oxidase and inability to utilise arginine, were
retained for further characterisation.
Characterisation by Polymerase Chain Reaction (PCR). All PCR
experiments were carried out on an Eppendorf Mastercycler®
Gradient using 20 ng of total DNA per reaction. The final reaction
volume was 30 μl including 10 μM of each primers and 1 unit of i‐Taq™ from INtRON Biotechnology Inc. The primers and the
programs were those published earlier (e.g. 2). For each
experiment, a negative control, in which the DNA solution was
replaced by water, and a positive control, in which the DNA was
that of a strain we knew would give a positive reaction, were
used. Of the 15 strains analysed, five gave a positive reaction
with primers specific to strains of genomospecies 1, which is
represented by P. syringae pv. syringae. None gave a positive
response with primers specific for strains of genomospecies 2,
which is represented by P. syringae pv. phaseolicola and P. syringae pv morsprunorum. None gave a positive response when
PCR protocols specific for P. syringae pv. papulans, P. syringae pv tagetis, P. syringae pv helianthi or P. syringae pv actinidiae were
used.
DISCUSSION
Strains of P. syringae were isolated from two different water
systems: the Waikato River, a complex system which includes
lakes and goes through some cultivated and non cultivated
lands, and the Whakapapanui Stream, which is fed by melt water
from Mount Ruapehu and does not cross cultivated lands. These
results and similar ones presented earlier (3) support the
hypothesis that the life history of P. syringae is linked to the
water cycle, as proposed by Morris et al. (3). In this scenario, rain
and melt water containing cells of P. syringae feed streams and
rivers that bring those cells of P. syringae in contact with wild
and cultivated plants. The subsequent multiplication of these
bacteria as pathogens or epiphytes provides a huge inoculum,
part of which might form aerosols that can be taken up by
clouds. The ability of some of these bacteria to induce ice
nucleation might help the formation of rain and/or snow and
explain the presence of these bacteria in rain and snow.
Although strains of P. syringae have been found in a river and a
stream fed by melt water, to complete the cycle we need to
demonstrate that those same strains are also found on or in
plants as epiphytes or as pathogens. The characterisation of the
strains of P. syringae isolated from New Zealand waterways
would allow this demonstration. The characterisation is
continuing, with more strains being analysed including strains
being isolated from different waterways, and with more
techniques being utilised including molecular techniques
different from PCR.
ACKNOWLEDGEMENTS
We thank The New Zealand Institute for Plant and Food
Research Limited for their support.
REFERENCES
1. Gardan L, Shafik H, Belouin S, Broch R, Grimont F, Grimont PDA
1999. DNA relatedness among the pathovars of Pseudomonas
syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). International Journal of Systematic Bacteriology. 49: 469–478. 2. Kerkoud M, Manceau C, Paulin J‐P 2002. Rapid diagnosis of
Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, by polymerase chain reaction using specifically designed hrpL gene primer Phytopathology 92: 1077–1083. 3. Morris CE, Sands DC, Vinatzer BA, Glaux C, Guilbaud C, Buffiere A,
Yan S, Dominguez H Thompson BM (2008) The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle. The International Society for Microbial Ecology Journal 1, 1–14. 4. Vanneste JL, Cornish DA, Yu J, Boyd RJ, Morris CE (2008) Isolation of
copper and streptomycin resistant phytopathogenic Pseudomonas
syringae from lakes and rivers in the Central North Island of New Zealand. New Zealand Plant Protection 61, 80–85.
Session
5C—Chemical
control
Evaluation of fungicides to manage brassica stem canker
B.H. Hall, L. Deland{ XE "Deland, L." }, B. Rawnsley, T. Barlow, C. Hitch and T.J. Wicks
South Australian Research and Development Institute, GPO Box 397, Adelaide, 5001, South Australia
INTRODUCTION
Leptosphaeria maculans and Rhizoctonia solani AG 2.1, 2.2 and 4
are the dominant soil borne fungal pathogens causing Brassica
stem canker (1). Brassica stem canker causes stem rot, resulting
in plant collapse before harvest or stem breakage during
harvest. Greenhouse trials were undertaken to evaluate pre‐
planting and post‐planting fungicide drenches for the control of R. solani AG 2.1, 2.2 and 4 and L. maculans.
MATERIALS AND METHODS
R. solani. Coco peat potting mix was inoculated with either AG
2.1, 2.2 or 4 by mixing in a 1L slurry of 12 macerated plates of 5– 7 day old cultures grown on potato dextrose agar (PDA). The
inoculated mix was placed into MK12 pots and incubated in the
greenhouse at 22°C for 7 days to allow the Rhizoctonia to
establish in the soil.
Six‐week‐old susceptible cauliflower speedlings (cv. Chaser) (2)
were immersed in a fungicide mix (Table 1) for 5 mins to ensure
the fungicide had permeated the soil and root matrix before
planting into the inoculated soil mix. Ten replicate plants were
used per treatment, with a water drench used as the control
treatment.
Plants were maintained in a greenhouse at 22°C and assessed
weekly for stem canker using a disease severity rating scale of 0– 100 where; 0 = healthy, 20 = superficial staining, 40 = canker
girdling ½ stem, 60 = canker girdling full stem, 80 = severe canker
(wilt) and 100 = plant death. The presence of R. solani in the pots
was confirmed 4–6 weeks after planting by baiting with
toothpicks, whereby toothpicks were placed in the pots for 24
hrs, washed and incubated on PDA (3).
L. maculans. Four‐‐week‐old cauliflower seedlings cv. Chaser
were planted into MK12 pots with coco peat. Six replicate plants
were treated with 30 ml each of fungicide drench (Table 2)
applied to the soil surface either two days before or two days
after mycelial plug inoculation (2). Plants were maintained and
assessed similarly to the R. solani trial.
RESULTS AND DISCUSSION
R. solani. All controls were infected with R. solani, AG 2.1 being
the most virulent and AG 2.2 the least virulent, with 100% and
70% of untreated plants infected respectively. None of the
fungicides effectively controlled the disease; however Amistar,
Cabrio, Maxim, Sumisclex and Jockey did reduce the severity
(Table 1). The different AG groups responded differently to the
fungicides, for example Rizolex at 0.4ml/L did not suppress AG
2.1 or 4, but was effective against AG 2.2. R. solani was detected
in soil from all treatments (data not presented).
L. maculans. The disease developed slowly, with symptoms not
showing on many plants until 10 weeks after inoculation. All
control plants were infected, and none of the treatments were
effective when applied after inoculation (Table 2). Maxim, Cabrio
and Rovral provided some suppression of disease when applied
before inoculation, and complete control was achieved with a
pre‐ plant drench of the higher rate of Amistar.
Table 1. Mean per cent severity of stem canker symptoms on plants 8
weeks after being drenched with a fungicide prior to planting into R.
solani inoculated soil.
Treatment Rate /L AG2.1 AG2.2 AG4
Untreated 74 24 46 Sumisclex 500 0.75ml 18 12 18 Rizolex liquid 0.2ml 78 14 42 Rizolex liquid 0.4ml 62 2 82 Jockey 1ml 10 6 26 Terrachlor 2g 52 16 24 Rovral Aquaflo 0.5ml 36 6 36 Rovral Aquaflo 1ml 30 6 34 Amistar 0.5ml 20 4 10 Amistar 1ml 16 10 14 Cabrio 0.4ml 16 12 22 Maxim 0.4ml 16 6 10 L.S.D (P=0.05) 14.5 12.7 17.0
Table 2. Per cent incidence (inc) and severity (sev) of stem canker
symptoms on plants drenched with fungicides and inoculated with L.
maculans before or after treatment.
Pre inoc.
drench Post inoc. drench
Treatment Rate /L Inc Sev Inc Sev
Untreated 100 70 100 27 Sumisclex 500 0.75ml 100 40 100 67 Rizolex liquid 0.2ml 67 17 83 27 Rizolex liquid 0.4ml 83 30 100 27 Jockey 1ml 83 27 100 24 Terrachlor 2g 100 20 100 30 Rovral Aquaflo 0.5ml 83 17 100 44 Rovral Aquaflo 1ml 17 3 100 23 Amistar 0.5ml 50 14 67 24 Amistar 1ml 0 0 67 20 Cabrio 0.4ml 33 7 100 27 Maxim 0.4ml 33 7 67 20 LSD (P=0.05) ‐ 16.2 ‐ 16.5 CONCLUSION
No fungicide treatments were effective in controlling stem
canker; however suppression was achieved with a pre planting
drench of either Amistar, Cabrio or Maxim.
ACKNOWLEDGEMENTS
This project was facilitated by HAL in partnership with AUSVEG
and was funded by the National Vegetable Levy. The Australian
Government provides matched funding for all HAL’s R&D
activities.
REFERENCES
1. Hitch C.J. et al (2006). Identifying the cause of brassica stem canker. 4th Australasian Soilborne Diseases Symposium, NZ, 2006. 2. Hall, B. et al (2009). Varietal resistance of cauliflower cultivars to
soilborne diseases Rhizoctonia solani and Leptosphaeria maculans. 5th Australasian Soilborne Diseases Symposium, NSW, 2009. 3. Paulitz TC & Schroeder KL (2005) A new method for the
Quantification of Rhizoctonia solani and R. oryzae from soil. Plant