Australasian Plant Pathol. (2013) 42:403–411 DOI 10.1007/s13313-013-0211-5
Pythium species associated with root rot complex in winter-grown parsnip and parsley crops in south eastern Australia J. E. Petkowski & R. F. de Boer & S. Norng & F. Thomson & E. J. Minchinton
Received: 30 July 2012 / Accepted: 12 March 2013 / Published online: 6 April 2013 # Australasian Plant Pathology Society Inc. 2013
Abstract Root rot of parsnip (Pastinaca sativa) and parsley (Petroselinum crispum) crops in south-eastern Australia is a disease complex, causing up to 80 % and 100 % yield losses, respectively. It is attributed to a range of fungi and oomycetes. We report on isolation and identification of eleven Pythium spp. from naturally infected roots of parsnip and parsley collected during systematic monthly surveys of commercial, winter-grown crops with prevailing root rots at Devon Meadows and Clyde, Victoria, Australia in 2009 to 2011. Pythium intermedium, P. ultimum var. ultimum, P. rostratifingens and species from the P. dissotocum complex were common to both hosts. Pythium irregulare, P. sylvaticum, P. camurandrum, P. vanterpoolii and P. tracheiphilum were isolated only from parsnip; and P. mastophorum and P. sulcatum only from parsley. This is the first report of P. camurandrum and P. rostratifingens in Australia and the first report of P. tracheiphilum and P. vanterpoolii from parsnip in Australia. Selected Pythium spp. tested for their pathogenicity in glasshouse conditions were pathogenic to both hosts. Pythium sulcatum, P. mastophorum and P. tracheiphilum caused severe root in 35 %, 53 % and 25 % of parsley seedlings, respectively. Pythium sulcatum and P. intermedium caused severe root in 50 % and 15 % of parsnip seedlings, J. E. Petkowski (*) : R. F. de Boer Department of Primary Industries, AgriBio, Centre for AgriBioscience, La Trobe University, 5 Ring Road, Bundoora 3086 VIC, Australia e-mail:
[email protected] E. J. Minchinton Department of Primary Industries, Biosecurity Victoria, Knoxfield Centre, Private Bag 15, Ferntree Gully DC 3156 VIC, Australia S. Norng : F. Thomson Department of Primary Industries, Future Farming Systems Research, Knoxfield Centre, Private Bag 15, Ferntree Gully DC 3156 VIC, Australia
respectively. In growth chamber studies, P. sulcatum was pathogenic to parsley seedlings at 10 °C, 18 °C and 24 °C. Keywords Oomycete . Root rot complex . Pathogenicity . Parsley . Parsnip
Introduction Root rot of parsley and parsnip crops in southeastern Australia is a disease complex, causing up to 100 % and 80 % yield losses, respectively (Minchinton et al. 2012). Symptoms range from minor root lesions to damping-off of seedlings and root rot in mature crops. In parsnip, the disease complex is also known as parsnip canker. In Australia, numerous fungal and oomycete species were isolated directly from diseased roots or from pear fruit bioassays of parsley-growing soils, including P. ultimum, P. sulcatum, P. acanthophoron, P. intermedium, P. irregulare, P. paroecandrum, the mycoparasite P. oligandrum, species from the P. diclinum and P. littorale groups, as well as unidentified species (Minchinton et al. 2006, 2007). Elsewhere, root rot and damping off in parsley has been attributed to P. paroecandrum in Northern Ireland (McCracken 1984a, b), P. mastophorum in Germany (Krober and Sauthoff 1999) and P. aphanidermatum in hydroponic crops in South Africa (Gull et al. 2004). Damping-off in parsley in the USA, has been associated with P. ultimum, P. irregulare and Rhizoctonia solani (Hershman et al. 1986) and P. debaryanum (De Zeeuw 1954), while in Belgium and Poland, it was associated with several fungi and Pythium (Nowicki 1997; Nawrocki and Mazur 2004). Parsnip canker, has been largely attributed to the fungi Itersonilia perplexans, Phoma spp., Mycocentrospora acerina, Streptomyces scabies and Cylindrocarpon destructans in the UK (Jones 1953; Channon 1956, 1965; Channon and Thomson 1981; Fox 2002), Phoma complanata in Canada
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(Cerkauskas 1985), and Itersonilia perplexans in the USA (Wilkinson 1952). The disease has not been associated with bacteria (Green and Hewlett 1950). Studies conducted by Minchinton et al. (2008) in Victoria, Australia concluded that parsnip canker can involve species from the genera Pythium, Fusarium, Rhizoctonia, Acremonium, Cylindrocarpon, Microdochium, Itersonilia and Phoma. Isolates from these genera caused severe lesions in pathogenicity tests on parsnip roots, but Rhizoctonia and Itersonilia only caused moderate symptoms. Little is known about the epidemiology of parsnip canker in relation to the phenological stage of crop development. Systematic crop monitoring, and identification of pathogens associated with distinct disease symptoms from seeding to harvest, was recommended by Minchinton et al. (2008) to precisely determine pathogen succession during crop development to target disease control methods. This work reports on the identification of Pythium spp. isolated from roots of winter-grown commercial parsley and parsnip crops in south eastern Australia in 2009, 2010 and 2011 and on the pathogenicity of selected isolates on seedlings of both hosts.
Materials and methods Plant sampling from trial sites Parsley and parsnip plants were systematically sampled in monthly disease surveys from untreated control plots of two parsley and five parsnip replicated field disease management trials in the market garden area south east of Melbourne, at Devon Meadows and Clyde (Table 1). The two parsley trials were directly seeded with parsley ‘Continental’ in three rows per bed on raised beds. The parsnip trials were directly seeded with the grower’s own seed in four rows per bed on raised beds. In all cases, crops were maintained by the growers. In the field trials, plants were sampled from each untreated control plot at approximately monthly intervals from May to November (a month before harvest), depending on the trial site and cropping season (Table 1). In the parsley trials, four plants were randomly sampled from each 1 m long section of bed at the ends of untreated control plots, giving a total of eight plants per plot. In the parsnip trials, two plants were randomly sampled from each 1 m long bed section at the ends of every untreated control plot, giving a total of four plants per plot. In parsley trials at harvest time, plants were sampled from the entire area of the untreated control plots, 12 plants in the 2010 trial and 11–17 plants in the 2011 trial. In the 2009 parsnip trials at harvest time, 36 and 64 plants were sampled from the entire area of each control plot at the Devon Meadows and Clyde trials respectively. At the 2010 and 2011 parsnip harvests, 20 plants were sampled from the entire area of the control plots from each trial.
J.E. Petkowski et al.
Isolations and identification of Pythium spp. from asymptomatic and symptomatic plants Pathogens were isolated from non-symptomatic and symptomatic roots of both hosts sampled during monthly surveys at each trial site. Sections of roots, approximately 2 mm long, including fine roots, were washed in sterile distilled water (SDW), blotted dry, plated onto water agar (WA) and incubated at room temperature (20 °C). After 2–3 days, cultures characteristic of Pythium were identified microscopically and hyphal tips transferred in 5 mm diameter plugs onto V8 juice agar or into 250 mL conical flasks with V8 juice broth. Morphological structures of Pythium spp. were confirmed microscopically, using standard keys (van der Plaats-Niterink 1981; Bala et al. 2010). Mycelia of 7 to 10 day old Pythium-like isolates were scraped from agar plates with a scalpel blade, or vacuum filtered on a Buchner funnel from liquid cultures, and stored in 1.5 mL centrifuge tubes at −20 °C for DNA extraction and identification. DNA extraction and ITS sequencing DNA was extracted from 57 Pythium-like isolates using FastDNA®SPIN for Soil Kit (MP Biomedicals, LLC) according to the manufacturer’s instructions. DNA concentration was measured using a Nano Drop® ND-100 spectrophotometer (BioLab Australia Pty Ltd). The ITS region, including the 5.8S gene of the Pythium isolates was amplified using primers UN-UP18S42 (5′-CGTAACAAG GTTTCCGTAGGTGAAC-3′) (Bakkeren et al. 2000) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al. 1990). DNA was amplified by polymerase chain reactions (PCR) in an Applied Biosystems Veriti Thermal Cycler. For both primer pairs, each reaction consisted of 0.2 μM of primer, 1 mM of dNTP, 5 μL of 1 × buffer (Invitrogen), 2 mM MgCl2, 1U of Platinum® TaqDNA polymerase (Invitrogen) and 2 μL of DNA (of a concentration of approximately 30 ng/μL) in a final volume of 50 μL. PCR conditions for DNA samples were as follows: initial denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 s, primer annealing at 50 °C for 30 s, and elongation at 72 °C for 1 min. The final extension step of 7 min at 72 °C was the same for both primers pairs. PCR products were resolved by electrophoresis on 2 % agarose gel stained with ethidium bromide. The products were then purified (QIAquick PCR purification Kit, Qiagen) according to the manufacturer’s instructions. Sequencing was done by Australian Genome Facility Ltd performed on an AB 3730xl automated sequencer (Applied Biosystems) after DNA labelling (sequencing) reaction of PCR products with relevant primers. The isolates were identified to the species level by conducting Basic Local Alignment Search Tool (BLAST) searches with the sequence data in GenBank.
Pythium species associated with root rot of parsnip and parsley
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Table 1 Site details of the market garden area south east of Melbourne where monthly disease surveys were conducted on parsley and parsnip roots sampled from untreated control plots in seven disease management trials in 2009, 2010 and 2011 Trial
Parsley 2010 Devon Meadows 2011 Clyde Parsnip 2009 Devon Meadows Clyde 2010 Devon Meadows Clyde 2011 Devon Meadows
Soil type
No. of replicate plots
Trial dates
No. of plants sampled per plot
Set up
Harvest
May to September
Harvest
Sand
5
20 April
6 September
8
12
Sand
7
1 June
28 October
8
11–17
Medium clay Sandy loam
6 8
8 April 8 April
29 October 21 October
4 4
36 64
Sandy loam Sandy loam
6 12
16 April 13 May
28 October 2 December
4 4
20 20
Sandy loam
12
8 April
24–25 October
4
20
Pathogenicity of Pythium spp. on parsley and parsnip roots Eight Pythium isolates were selected for pathogenicity testing on parsley and parsnip seedlings (Table 2). These included species that had not been reported before on either host, in Australia and those species previously reported from Apiaceae and isolated relatively frequently in our survey. In addition, an isolate of P. mastphorum was also tested on parsley, a host on which this species had been reported overseas (Krober and Sauthoff 1999). This isolate was not included in the pathogenicity tests on parsnip because it was not isolated from this host in our trials. Inocula were prepared from mycelia stored on WA plugs in sterile distilled water in McCartney bottles at room temperature. Single plugs were plated and grown initially for 2 days on WA. Mycelial tips were then subcultured onto V8 juice agar and incubated at room temperature. Plugs (1 cm diameter) were cut from the edge of 5 day old Pythium cultures and used for inoculations in each of the three experiments conducted in growth cabinets and the glasshouse. Pathogenicity of Pythium spp. on parsnip Parsnip ‘Melbourne White Skin’ seedlings were raised in seed raising mix (Debco™ Debco Pty. Ltd. Tyabb, Victoria), in 29×35 cm plastic trays on a glasshouse bench on 20th April 2012. Glasshouse day/night temperatures were set at 25 °C and 17 °C, respectively. Seedlings were irrigated twice a day for 1 min at 6 am and 2 pm by overhead sprinklers and fertilised weekly with 5 g/10 L solution of
Aquasol™ fertiliser. Four three-week-old seedlings (2–3 true leaf stage) were transplanted into 10 cm diameter plastic pots (45 pots in total). Four weeks after transplanting, pots were inoculated with eight 10 mm diameter agar plugs of one of eight Pythium spp. or left uninoculated as a control. Pots were arranged on a glasshouse bench in five blocks of nine randomised treatments (eight Pythium spp. and an uninoculated control). Seedlings were assessed for disease symptoms 5 weeks after inoculation, as described below. Pathogenicity of Pythium spp. on parsley Parsley ‘Italian Plain Leaf’ was seeded into seed raising mix (Debco™ Debco Pty. Ltd. Tyabb, Victoria) in 7.5 cm diameter plastic pots on a glasshouse bench on 10th May 2012. Glasshouse conditions, irrigation and fertiliser applications were as described above. Five weeks after seeding (5 plants/pot, 4 pots/rep), each pot was inoculated with six V8 juice agar plugs with one of nine Pythium spp. or left uninoculated as a control. Pots were arranged in four blocks of 10 randomised treatments (nine Pythium spp. and an uninoculated control). Seedlings were assessed for disease symptoms 5 weeks after inoculation, as described below. Pathogenicity of P. sulcatum on parsley at different temperatures Parsley ‘Italian Plain Leaf’ was seeded into seed raising mix (Debco™ Debco Pty. Ltd. Tyabb, Victoria) in plastic multicell trays (144 cells per tray) on a glasshouse bench
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Table 2 Pythium spp. isolated from parsley and parsnip roots in field surveys selected for pathogenicity testing, their hosts, locations, collection dates and relevant reference numbers VPRI Acc. No.
GenBank Acc.
Pythium sp. (ITS sequence)
Isolation date
Host
Symptom
42087 42088 42090 42085 42086 42108 42082 42089 42083
KC689900 KC689901 KC689904 KC689905 KC689903 KC689902 KC689906 KC689899 KC689907
P. dissotocum complex P. dissotocum complex P. sulcatum P. tracheiphilum P. mastophorum P. intermedium P. ultimum var. ultimum P. camurandrum P. rostratifingens
24/06/10 15/06/10 16/07/10 22/07/11 15/06/10 19/08/11 18/05/09 22/07/09 18/05/09
Parsnip Parsley Parsley Parsnip Parsley Parsley Parsnip Parsnip Parsnip
Brown lesion on lower tap root Soft rot of tap root Lesions on tap and fine roots Crown with rotten leaf bases Brown sunken lesion on tap root Pruned lateral roots Rotten collar Rotten collar Rotten collar
on 25th May 2010. Glasshouse conditions, irrigation and fertiliser applications were as for the pathogenicity test on parsnip. Four-week-old seedlings at the 4–6 true leaf stage were transplanted into a 7.5 cm diameter plastic pot (one seedling per pot), filled with seed raising mix. All pots were fertilised with 1 g of slow-release complete fertiliser Nitrophoska ™ (Brunnings) and transferred to each of three growth cabinets set at constant 10 °C, 18 °C and 24 °C. Each cabinet was set at 12 h light/dark with a light intensity of 270 μE mol m−2 s−1 and relative humidity of 70 %. Seedlings were watered to saturation every second day. After 3 weeks, each of twelve pots in each cabinet were inoculated with six agar plugs taken from P. sulcatum culture (isolated from parsley by Minchinton et al. (2006)) placed under a thin layer of seedling mix. The remaining twelve pots in each growth cabinet served as uninoculated controls. All 24 pots in each of the cabinets were flooded (full water holding capacity of the seedling mix) for 24 h after inoculation to initiate pathogen zoospore release. Seedlings were assessed for disease symptoms 2 weeks after inoculation, as described below. Disease assessment At harvest, plants were assigned one of four categories of root disease severity (0: healthy, 1: minor lesions (specks), 2: lesion(s) on tap, lateral and fine roots, and 3: deep lesions and pruning of tap root, lateral and fine roots) and disease incidence was calculated as a percentage of plants with these symptoms. The total fresh weight of each plant (shoots and roots) was recorded for each inoculated and control plant to determine the effect of pathogenicity on plant growth. Sections of infected host roots were rinsed in SDW and plated on WA and examined microscopically for emergence of hyphae with morphological characteristics of the Pythium spp. tested to confirm Koch’s postulates.
Statistical analysis Disease severity data (tap roots, lateral roots and fine roots) from pathogenicity tests of nine species on parsley and eight species on parsnip were analysed using ordinal regression appropriate for disease score data (Coe 2002). Data of the number of plants with root rot severity rating scores (tap, lateral and fine roots) 0 and 1 combined (slight), 2 (moderate) and 3 (severe root damage) were modelled as a cumulative multinomial distribution with a logit link. An analysis of deviance analogous to an analysis of variance (ANOVA) was performed. Treatments (pathogens) were compared with the uninoculated control by means of standard errors of difference (sed). Plant fresh weight data for parsnip and parsley from pathogenicity tests data were analysed using analysis of variance (ANOVA) appropriate for a randomised complete block design (RCBD) with a permutation test using the default 9999 iterations. The treatment structure was specified in GenStat as Treatment (pathogens) and the block structure was specified as Rep/Plant. Histograms of residuals and graphs of residuals versus fitted values were used to check distributional normality assumptions. All statistical analyses were conducted in GenStat 14.1 (Payne et al. 2011).
Results Isolation and identification of Pythium spp. from plants in trial sites Eleven species or species complexes were identified by ITS sequence data and macroscopic confirmation of morphological structures, from a total of 57 Pythium isolates collected from non-symptomatic and symptomatic parsley and parsnip roots sampled during crop surveys in 2009, 2010 and 2011 (Table 3). Six isolates (four from parsnip and two from parsley) were
Pythium species associated with root rot of parsnip and parsley Table 3 Pythium spp. isolated from parsnip and parsley roots sampled from untreated control plots in field trials in the market gardens south east of Melbourne, Victoria, Australia identified from ITS rDNA sequence data. Species are listed according to the phylogeny of Lévesque and de Cock (2004)
a
Members of clade B2 (Lévesque and de Cock 2004) including P. dissotocum, P. coloratum, P. lutarium, which have identical ITS sequences
b
Pythium spp. not identified to species level
Identified Pythium spp.
Clade B P. dissotocum complexa P. sulcatum P. tracheiphilumd P. vanterpooliid Clade E P. camurandrumc P. rostratifingensc Clade F P. intermedium P. irregulare P. sylvaticum Clade I P. ultimum var. ultimum Clade J
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No of isolates from host
Commonly reported hosts
Parsnip
Parsley
8 0 2 1
2 1 0 0
Apiaceae (carrot, parsley) Apiaceae (carrot, parsley) Lettuce Grasses
2 2
0 1
Soil Leaf litter, soil under apple tree, corn, Brassica, Triticum, Medicago
7 2 3
2 0 0
Apiaceae (carrot, parsley) Apiaceae (carrot, parsley) Apiaceae (carrot, parsley)
4
2
Apiaceae (carrot, parsley) Apiaceae (parsley), soil under pine plantations
c
Not reported in Australia on any host
P. mastophorumc
0
1
d Not reported in Australia on parsnip or parsley
P. sppb
4
2
confirmed to be the genus Pythium but could not be identified to species level using sequence data. Nine Pythium spp. were identified from 35 sequences and six from eleven sequences of rDNA ITS (ITS1, 5.8S, and ITS2) from the parsnip and parsley isolates, respectively. Pythium ultimum var. ultimum, P. intermedium and P. rostratifingens and species belonging to the P. dissotocum complex were common to both hosts. Pythium sulcatum and P. mastophorum were isolated only from parsley and P. irregulare, P. sylvaticum, P. tracheiphilum, P. vanterpoolii and P. camurandrum were isolated only from parsnip. Pythium rostratifingens and P. camurandurum have not been previously reported in Australia, while P. tracheiphilum and P. vanterpoolii have not been recorded on parsley or parsnip in Australia. Based on molecular phylogeny and taxonomy of the genus Pythium (Lévesque and de Cock 2004), the species identified in this study belong to five phylogenetic clades: B, E, F, I, and J. Sequences of the P. dissotocum complex are identical to those of P. dissotocum, P. lutarium and P. coloratum. These three species are also morphologically similar (Lévesque and de Cock 2004), and are therefore named here as a P. dissotocum complex. Eight and nine species were selected for pathogenicity testing on parsnip and parsley seedlings, respectively (Table 2). Pathogenicity of Pythium spp. on parsnip The results of the pathogenicity tests presented in Table 4 show that all isolates of Pythium tested caused root damage
to parsnip seedlings with disease incidence ranging from an average of 20 to 100 % of plants affected and average severity scores ranging from 0.1 to 2.3 (scale 0–3). Symptoms of root rot were not found in the roots of the uninoculated control plants. Only P. sulcatum and P. intermedium caused severe root damage (average root rot severity rating of 3 on tap, lateral or fine roots) with 50 % and 15 %, of plants affected, respectively. The analysis of deviance (an output from ordinal regression in GenStat) of the log odds ratios of disease score ratios is interpreted similarly to the analysis of variance, where the deviance ratio (deviance of effects/residual deviance) is computed and then compared with a chi squared distribution to determine the significance of an effect. The regression (treatment effect) was shown to be significant (P
