title / Risk assessment of recombinant progeny of Phytophthora infestans: pathogenicity, fungicide sensitivity and ecotypic variation
/ DEFRA
project code / CSA5700
Department for Environment, Food and Rural Affairs CSG 15
Research and Development
Final Project Report
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Project title / Risk assessment of recombinant progeny of Phytophthora infestans: pathogenicity, fungicide sensitivity and ecotypic variation
DEFRA project code / CSA5700
Contractor organisation and location / School of Biological Sciences
University of Wales,
Bangor
Gwynedd
LL57 2UW
U.K.
Total DEFRA project costs / £ 115,272.06
Project start date / 01/05/01 / Project end date / 01/05/03
Executive summary (maximum 2 sides A4)
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CSG 15 (Rev. 6/02) 2
Projecttitle / Risk assessment of recombinant progeny of Phytophthora infestans: pathogenicity, fungicide sensitivity and ecotypic variation
/ DEFRA
project code / CSA5700
Summary
Twenty eight in vitro matings between A1 x A2 isolates, representing the two most common UK phenotypes of the potato late blight pathogen, Phytophthora infestans, produced oospores in all cases, but only one cross yielded sufficient hybrid single oospore progeny for characterisation studies. The ratio of A1:A2 progeny was 1.8:1, whilst almost two thirds were non pathogenic. Six of 28 pathogenic progeny were more aggressive than either parent with shorter latent period and faster lesion expansion on three potato cultivars with low, medium and high non race specific resistance to late blight. Parents and progeny were sensitive in vitro to cyazofamid and azoxystrobin but showed a range of response to metalaxyl, propamocarb and fluazinam. Although both parents were sensitive to the latter fungicide, a quarter of progeny exhibited in vitro resistance (>50% growth of control at 10ppm a.i.).
Seven North American isolates belonging to the US8 clonal lineage responded in the same way as UK isolates to the five fungicides. US8 isolates were more aggressive than UK isolates on inoculated leaf discs of two out of three potato cultivars and produced putative, but uncharacterized oosporic progeny in matings with a common UK clonal A1 mating type.
Inoculation of pot grown whole plants (cv. Maris Piper) at 12, 17 and 25 oC and saturated relative humidity with parental and recombinant UK isolates, US8 isolates and “old” displaced population UK isolates gave variable results depending on temperature. Although some single oospore progeny of the UK x UK mating were more aggressive than parental isolates it was concluded that established forecasting schemes using temperature criteria would not be compromised but novel schemes using response surface criteria may require reevaluation.
CSG 15 (Rev. 6/02) 2
Projecttitle / Risk assessment of recombinant progeny of Phytophthora infestans: pathogenicity, fungicide sensitivity and ecotypic variation
/ DEFRA
project code / CSA5700
Scientific report (maximum 20 sides A4)
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CSG 15 (Rev. 6/02) 2
Projecttitle / Risk assessment of recombinant progeny of Phytophthora infestans: pathogenicity, fungicide sensitivity and ecotypic variation
/ DEFRA
project code / CSA5700
Introduction
Late blight, caused by the oomycete Phytophthora infestans, remains a devastating disease of the potato crop. It is estimated to cost the industry US$3 billion annually in lost production and fungicides (1), on which growers are heavily dependent for disease control. Vegetative sporangia form the main source of inoculum. For sexual reproduction to occur, both A1 and A2 mating types usually must be present, producing oospores. Oospores are both a potential source of inoculum (2, 3) and also, through sexual recombination, of novel genotypes (4, 5). Both mating types have only been present together in Europe since the discovery of the A2 type in the 1980s (6, 7). There is now evidence of sexual reproduction in parts of Europe and North America (8, 9, 10, 11) where a genetically diverse population is associated with increased aggressiveness, tolerance to fungicides and greater difficulty in disease control (4, 5, 12, 13, 14). However, the A2 mating type so far remains rare in the UK, accounting for 5% of the population annually over a 6 year period from 1993 (15).
There are three aspects to this research programme; i) the potential impact of sexual reproduction on the UK population (to be assessed by generating and characterising sexual progeny), ii) the effect of any introduction of foreign isolates and iii) the need to update current blight forecasting models, some of which still depend on data from strains that were displaced by a more aggressive population in the 1980s (12). The US8 strain was chosen as an example of the potential effect of an accidental introduction of foreign isolates. US8 is a common phenotype on potatoes in the USA (11). It is highly aggressive, resistant to metalaxyl and an A2 mating type (5).
Material & Methods
Selection of isolates
Isolates representing common strains found in the UK were crossed to generate oospores. Parents were chosen on the basis of the most recent survey available, documenting genetic diversity in the P. infestans population (15). The most common A1 strain in the UK is designated RF39 on the basis of RG57 fingerprints. The RG57 nuclear DNA probe has been used in studies of the population genetics of P. infestans for some years (16, 17). The most common A2 was designated RF40. Parents were chosen from these two genotypes. The US8 strains were taken from the culture collection held at Bangor, where they had been stored in liquid nitrogen. All cultures were then routinely maintained on Rye A agar, at 18oC in darkness.
Generation of progeny
Twenty-eight matings were performed in the laboratory between isolates of UK origin (RF39 x RF40). Crosses were made between nine A1 and three A2 isolates, representing RF39 and RF40 genotypes respectively, collected from across England and Wales. UK isolates used and their origins are shown in Table 1. A further 27 matings were performed between nine RF39 (A1) isolates and three US8 (A2) (Table 2) isolates.
To achieve matings, strips of agar (3cm x 0.5cm) from actively growing A1 and A2 cultures were placed parallel to each other in Petri dishes containing either Rye A or V8 agar. Dishes were incubated in darkness at 18oC until oospores had formed (~2 weeks) and then transferred to white fluorescent light at room temperature until processed (1 week). Agar containing oospores was macerated in a glass homogeniser, suspended in ~2ml sterile water, filtered with 20µm pore nylon mesh (3) and re-suspended in 10ml water. The suspension was then treated with either an enzyme product (3, 18) or potassium permanganate (19) to kill asexual structures and trigger oospore germination. Germinating oospores were transferred to Rye A agar. Each cross was repeated 2-4 times. Mating type of progeny was determined (20) by dual culture with known A1 and A2 types on Rye A agar and assessing oospore production after 1 week.
After this method of generating progeny proved unsuccessful with US8 x UK crosses, the following modifications were made. After agar containing oospores was excised and macerated, it was suspended in 2-3ml water and chilled to 8oC overnight, then frozen at -20oC for at least an hour to kill asexual structures (21). The suspension was then thawed and spread thinly over the abaxial surface of detached potato leaves (cvs. Home Guard and Maris Piper) with a glass rod. The leaves were laid on damp tissue paper in sealed plastic trays and incubated in light at 18oC. Developing lesions were observed daily for 7 days, cut out with a scalpel and sub-cultured onto fresh leaves or agar. Controls (from single mating type cultures, i.e. sporangia and hyphal fragments but no oospores) resulted in no lesions over the same period, suggesting a complete kill of asexual structures. Three crosses between US8 (A2) isolates pi.174.2, pi.169.1, pi.161 and 96.36.1 (A1) were performed in this way.
Fungicide sensitivity
Progeny were tested against each of five fungicides; metalaxyl, propamocarb, fluazinam, cyazofamid and azoxystrobin. These include fungicides commonly used in control of blight as well as emerging products. Fluazinam (marketed as Shirlan) is the fungicide formulation most widely applied against blight in Britain, with 60% of the potato acreage given at least one application (22). Metalaxyl, a phenylamide, has been widely used against blight from the late 1970s. Recently, mefenoxam, an enantiomer of metalaxyl is employed as a systemic fungicide, combined with a protectant such as mancozeb (e.g. Fubol Gold WG). Propamocarb hydrochloride, another systemic, is also frequently combined with mancozeb. Cyazofamid is a newly developed cyanoimidazole, which acts to disrupt the activity of the zoospore stage of the lifecycle (23) while azoxystrobin inhibits mitochondrial respiration. All the fungicides used in experiments are registered for use against late blight in this country, with the exception of azoxystrobin, which currently is used against late blight in the USA. Fluazinam, azoxystrobin and cyazofamid are most effective as protectants. For summary of fungicide formulations, see Table 3.
All fungicides were added to rye agar at concentrations of 10 ppm active ingredient (20, 24). When amending agar with propamocarb, Rye B agar was used in preference to Rye A to avoid sequesterization by plant sterols. These in vitro tests were comparable to in vivo studies described by Bardsley et al. (25).
Table 1. Isolates of UK origin used in matings
ISOLATE / A1/A2 / ORIGIN96.70* / 2 / Anglesey
98.81.4 / 2 / N. Yorkshire
98.92.1.3 / 2 / Cheshire
98.30.9 / 1 / Lincolnshire
98.29.1 / 1 / Norfolk
98.88.1 / 1 / N. Yorkshire
98.70.12 / 1 / N. Yorkshire
98.3 / 1 / Anglesey
98.105.1 / 1 / Carmarthenshire
98.103.1 / 1 / Lincolnshire
98.78.6 / 1 / Cheshire
96.36.1* / 1 / Hampshire
* parents of the successful UK cross
Table 2. US8 isolates used in matings and characterisation
Isolate / Mating type / OriginPi.174.2* / 2 / Nebraska
Pi.161* / 2 / North Dakota
US97007* / 2 / New York
Pi.163 / 2 / North Dakota
Pi.169.1 / 2 / North Dakota
US970011 / 2 / Wyoming
Pi.160.2 / 2 / North Dakota
* Used in matings with isolate 96.36.1 of UK origin
Table 3. Fungicides
FUNGICIDE TRADE NAME / ACTIVE INGREDIENT(S) / DISTRIBUTOR / RECOMMENDED DOSE RATEFubol Gold WG / Metalaxyl-M1/ mancozeb / Syngenta / 1.9 kg granules / ha
Previcur / Tattoo / Propamocarb/ mancozeb / Aventis / 4 L / ha
Shirlan / Fluazinam / Syngenta / 0.3 L / ha
Ranman Twinpack / Cyazofamid / BASF / 0.2 L / ha Pack A, 0.15 L / ha Pack B2
Amistar / Azoxystrobin / Syngenta / 1 L / ha
1Pure metalaxyl used in tests for metalaxyl sensitivity. Only commercial fungicide formulations were available for remaining tests, so agar was amended with these (24). 2Pack A fungicide, Pack B adjuvant.
Pathogenicity
Pathogenicity was tested initially by inoculating leaflets from the highly susceptible potato cv. Home Guard with a sporangial solution from each of the in vitro cultured progeny from the 96.36.1 x 96.70 cross. Sterile water (~2ml) was added to Petri dishes containing the growing culture, the liquid removed by pipette into glass universal bottles and 50µl drops of the sporangial suspension placed on the abaxial surface of each leaflet. Three replicate leaflets per isolate were used in each of the three experiments, laid in a random design on damp filter paper in sealed trays to ensure high humidity and incubated at 18oC, in the optimum temperature range for growth of P. infestans.
Aggressiveness
Aggressiveness of progeny from the single UK mating was tested on 3-7 potato cultivars. Several components of aggressiveness are described here, including the length of the latent period (time from inoculation to sporulation), lesion size and rate of lesion expansion. Seven cultivars were initially considered but the number of cultivars screened was subsequently reduced to three; Home Guard (highly susceptible with a foliar blight rating of 3), Maris Piper (moderately susceptible, rating 5 and the most widely grown potato in Britain) and Sante (resistant, rating 7). Four replicate leaf discs (25mm diameter) for each isolate / treatment combination were placed in a random design on damp filter paper, inoculated with a 20µl sporangial suspension and incubated as for pathogenicity tests (10). The suspension was standardised (to 1.5 – 3 x 104) to ensure broadly equal numbers of sporangia were applied to each leaf disc and the suspension diluted as required. Presence / absence of sporulation and lesion size were measured daily over seven days. Results are summarised in Tables 4-6. Analysis of variance was performed (P ≤ 0.05) Lesion expansion was measured daily, but to summarise these data, mean time (days) for the lesion to cover the entire 25mm disc was calculated.
Each of the two UK parents from the most successful mating was tested for aggressiveness. Pathogenic progeny from that mating were also tested, as were seven US8 isolates. Fifteen possible progeny from US8 x RF39 matings (four isolates from both pi.174.2 x 96.36.1 and pi.169.1 x 96.36.1 crosses, seven from pi.161 x 96.36.1) were also included were evaluated only on cv. Maris Piper.
Virulence
Virulence refers to the ability of an isolate to infect potato leaves from plants containing specific resistance genes (R-genes) and is unrelated to aggressiveness as outlined above. In P. infestans a single gene (avr) enables isolates to overcome the opposing R-gene in the host (gene-for-gene theory, for details see Birch et al. 2001, ref. 26). A subset (15 isolates) of pathogenic progeny from the UK cross and the parents (96.36.1 and 96.70) were used to infect leaves representing the series of available R-genes, from R1 to R11, along with the cultivar Home Guard, which does not contain any known R-genes. The progeny tested were designated #6, #9, #11, #13, #17, #27, #28, #29, #35, #41, #55, #64, #66, #69 and #70; and using three replicate leaflets, placed on damp paper as above, inoculated and infection scored after 7 days incubation at 18oC.