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Sources, survival and management of Pestalotiopsis sydowiana on Calluna vulgaris nurseries
Crop Protection 20 (2001) 591}597
Sources, survival and management of Pestalotiopsis sydowiana on Calluna vulgaris nurseries M.P. McQuilken*, K.E. Hopkins Department of Plant Biology, The Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK Received 9 October 2000; received in revised form 12 January 2001; accepted 12 January 2001
Abstract Sources of Pestalotiopsis sydowiana were determined on commercial nurseries producing Calluna vulgaris throughout the UK. The pathogen was isolated from diseased stock plants, nursery soils, used growing media, propagation trays and #oor covering and dust collected from greenhouse walkways. P. sydowiana was not isolated from new or unused materials. Infection of cuttings of C. vulgaris was initiated from a range of contaminated or infected sources. The pathogen was able to survive on infected plant debris for up to 1 year to initiate new infections. Greenhouse trials were conducted to evaluate the e!ects of disease management methods (irrigation, tray disinfection and fungicide application) on control during the propagation of C. vulgaris. Disease incidence and foliar browning caused by P. sydowiana were less on cuttings watered by sub-irrigation compared with watering from overhead. Single and combined treatments of tray disinfection (hydrogen peroxide/peracetic acid) and a three-spray programme of prochloraz to the foliage of cuttings signi"cantly reduced disease incidence and foliar browning compared to dipping trays in water. The combined tray disinfection and fungicide programme signi"cantly reduced disease incidence but not foliar browning, compared to tray disinfection or fungicide application alone. Fungicide application alone was better than disinfection. The importance of these "ndings for the integrated control of P. sydowiana on ericaceous plant nurseries is discussed. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pestalotiopsis sydowiana; Calluna vulgaris; Inoculum sources; Survival; Irrigation; Disinfectants; Fungicides
1. Introduction Previous studies on the biology and control of diseases in crops of Calluna vulgaris (L.) Hull and other ericaceous crops have focused on the pathogens Rhizoctonia, Phytophthora and Pythium spp. (Rutherford et al., 1989; Backhaus and Leopold, 1990; Harig and Backhaus, 1990; Litterick and Holmes, 1990; Litterick et al., 1995a, b; Litterick and McQuilken, 1998). Advances in the technology of production systems and increased knowledge of pathogen biology have led to improved control of these pathogens. However, growers of C. vulgaris continue to report heavy crop losses due to root and foliar diseases. During recent seasons, an increasing number of damaging infections of C. vulgaris, caused by Pestalotiopsis spp., have been found on plant samples submitted by growers
* Corresponding author. Fax: #44-1292-525314. E-mail address: [email protected] (M.P. McQuilken). Present address: Crop Protection Compendium, CAB International, Wallingford OX10 8DE, UK.
to the SAC Crop Health Centre. Serious damage has been seen on cuttings, potted-on-plants and stock plants. The symptoms are a necrosis and death of the foliage, with extensive acervuli production on a!ected plant parts (Hopkins, 1996). Sometimes, a general rotting of the root and stem base may also occur (Hopkins, 1996; Hopkins and McQuilken, 1997, 2000). Recent studies have shown that P. sydowiana is the predominant species of Pestalotiopsis associated with C. vulgaris as well as other ericaceous plants in the UK (Hopkins and McQuilken, 2000). Pathogenicity tests and host range studies have also indicated that isolates of P. sydowiana are not host speci"c and they can infect a wide range of hardy ornamental species (Hopkins and McQuilken, 2000). In view of the exchange of ericaceous plants and other hardy ornamentals between the UK and other European countries, it is likely that P. sydowiana may have become established throughout most Northern European countries. It is clear that P. sydowiana poses a real threat to the production of C. vulgaris by causing plant losses, reduced plant quality and disruption of production schedules;
0261-2194/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 2 8 - X
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severe disease symptoms often cause the plants to be unsaleable (Hopkins, 1996; McQuilken et al., 1999). Extensive use of polythene tunnels, fertilizers, controlled irrigation and large-scale monoculture of C. vulgaris, particularly in Scotland has created ideal conditions for the development of the disease (Hopkins and McQuilken, 1997). As P. sydowiana is di$cult to eliminate completely once it is introduced into nurseries, disease control may be more e!ective if based on a knowledge of the potential sources and survival of the pathogen. However, at present, there is very little such information available on P. sydowiana. The objectives of this study were to (i) identify sources of initial inoculum of P. sydowiana on nurseries producing C. vulgaris, and to assess whether infection by the pathogen could be initiated from such inoculum sources; (ii) monitor the survival of the pathogen on infected plant material; (iii) evaluate the e!ects of various disease management methods (irrigation, tray disinfection and fungicide application) on control. A brief preliminary report has already been published (McQuilken et al., 1999).
2. Materials and methods 2.1. Identixcation of inoculum sources To determine if stock plants of C. vulgaris were infected with P. sydowiana, samples with foliar browning, root and stem-base rots were collected from several nurseries in the UK. Diseased plant tissue (3}5 mm was surface sterilised in 10% (v/v) sodium hypochlorite for 5}10 s and rinsed in three changes of sterile distilled water. Surfacesterilised segments of tissue were blot-dried on sterile "lter paper and plated on Oxoid potato dextrose agar (PDA; Unipath, Basingstoke, Hants., UK) modi"ed to contain 100 g ml\ of erythromycin and streptomycin (PDAES). Plates were incubated at 203C, and after 3}5 days, mycelia typical of P. sydowiana were transferred to single 9 cm diameter Petri dishes of PDAES. Identi"cation of isolates of P. sydowiana was made based on morphological and cultural characteristics (Sutton, 1980). Samples of soil, used ericaceous plant growing medium, growing medium debris removed from used propagation trays and #oor covering, and dust swept from glasshouse walkways from two nurseries were tested for the presence of P. sydowiana. Samples were air-dried, sieved through a 2 mm sieve and 10 cm of each was shaken in 90 ml of sterile 0.01% agar on a wrist action shaker at low speed for 10 min. After standing for a further 20 min, a dilution series was made and 0.5 ml samples of the dilutions were spread-plated onto PDAES containing Triton X-100 (2 ml l\; BDH Laboratory Supplies, Poole, UK). Colonies of P. sydowiana were identi"ed after 7}10 days growth at 203C.
A greenhouse trial was conducted to assess whether infection of cuttings of C. vulgaris (cv. Kinlochruel) by P. sydowiana could be initiated from a range of potential inoculum sources. Cuttings obtained from disease-free 3-year-old stock plants were struck (70 per tray) into propagation medium (80 : 20 "ne peat : super "ne perlite; Bulrush Peat Company Ltd., Bellagly, N. Ireland) in 70-cell plastic plug trays (28;16 cm with 70;10 ml individual plugs). These trays were used in all subsequent trials. Preliminary trials indicated that prior wounding of plant tissue by dusting lightly with carborundum was necessary to establish infection of cuttings (Hopkins and McQuilken, 2000). Treatments included: (1) the incorporation of pathogen-infested propagation medium into trays of uninfested medium (1:10 v/v); (2) sprinkling samples (approximately 3 g per replicate tray) of pathogeninfested dust, collected from glasshouse #oors and walkways, over trays immediately before striking; (3) striking two pathogen-infected cuttings into the trays (two centre rows) of healthy cuttings; (4) dirty, used trays contaminated with the pathogen; (5) new, unused trays placed on Mypex (LBS Group, Colne, Lancs., UK) #oor covering contaminated with the pathogen. Appropriate control treatments were also set up using uncontaminated materials. Trays were placed under low polythene tunnels within a greenhouse maintained at 12}243C, and watered weekly by overhead irrigation. After 8 and 12 weeks, individual cuttings were assessed for incidence of Pestalotiopsis (presence of black spore masses) and foliar browning using a percentage browning scale of 0}100, where 0"no foliar browning and 100"foliage totally brown. To con"rm infection by P. sydowiana after 12 weeks, small pieces of diseased tissue were plated on PDAES and observed for colonies typical of the pathogen. 2.2. Survival on infected cuttings Shoots of cuttings infected with P. sydowiana were air-dried and stored in sealed plastic boxes (16;28;5 cm) at 183C in the dark. The ability of P. sydowiana to grow from stored cuttings was assessed after 0, 3, 6 and 12 months by plating 3}5 mm segments (4 per dish) of four cuttings on PDAES in 9 cm diameter Petri dishes. After 7}10 days of incubation at 203C, the number of cuttings giving rise to P. sydowiana was counted. The ability of P. sydowiana-infected plant material (cuttings) to initiate infection after each storage period was also evaluated by conducting a small-scale glasshouse trial. Four cuttings were removed from storage, cut into segments and sprinkled over the surface of "ve replicate plug trays containing uninfested propagation medium, prior to striking 70 pathogen-free cuttings (cv. Bognie) per tray. Trays of cuttings were wounded, placed in a glasshouse under low polythene tunnels, watered,
M.P. McQuilken, K.E. Hopkins / Crop Protection 20 (2001) 591}597
and assessed for disease incidence and severity as described before. Controls consisted of pathogen-free cuttings struck into uninfested propagation medium. 2.3. Ewect of irrigation A greenhouse trial was conducted to investigate the e!ect of di!erent irrigation methods on the incidence and severity of Pestalotiopsis. Cuttings (cv. Alba Praecox) were struck into plug trays as described before, except that two of the cuttings in the central two rows of each tray were infected with P. sydowiana as inoculum. Trays were placed on capillary sand-beds (for sub-irrigation) or Mypex (for weekly overhead watering) under low polythene tunnels within the glasshouse. Trays of cuttings were wounded, and assessed subsequently for disease incidence and severity as described before. 2.4. Ewect of tray disinfection and fungicide application A greenhouse trial was conducted to evaluate tray disinfection alone and in combination with a fungicide applied to cuttings on the incidence and severity of Pestalotiopsis. The fungicide, prochloraz, was selected as it is recommended for control of Pestalotiopsis on ericaceous plants, and gave good control against the pathogen on C. vulgaris in previous trials (McQuilken et al., 1997). To obtain trays contaminated with P. sydowiana, wounded cuttings were grown in new, unused trays and arti"cially inoculated by brushing individual cuttings with a spore suspension (1}3;10 conidia ml\). The spore suspension was prepared by #ooding 21-day-old PDA Petri dish cultures with sterile distilled water and gently scraping the colony surface with a sterile bent glass rod. After 12 weeks, infected cuttings were removed and contaminated trays were used in the trial. There were "ve treatments: (1) control, trays dipped in water for 1 h; (2) trays dipped in hydrogen peroxide (H O )#peracetic acid (CH CO.OOH) (Jet 5, 23%/5% s.c., Hortichem Ltd, Amesbury, Wilts., UK) for 1 h; (3) a three-spray programme of prochloraz (Octave, 46% w/w w.p., Scotts UK Professional, Ipswich, UK) at 0.46 g a.i. l\ applied at striking and at 14 day intervals thereafter; (4) combined treatments of 2 and 3; and (5) new, unused trays dipped in water for 1 h. The recommended rate of Jet 5 in water was used (1 : 125 v/v), and loose growing media and crop debris were removed by shaking prior to dipping. Following treatment, all trays were rinsed in water, allowed to dry, re"lled with propagation media and restruck with cuttings (cv. Alba Praecox). The fungicide sprays were applied to cuttings to run o!, using a Mardrive Precision sprayer at 300 kPa. Trays of cuttings were wounded, and assessed subsequently for disease incidence and severity as described before.
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2.5. Statistical analyses All glasshouse trials were arranged in randomised complete block designs with "ve replicates for each treatment, and repeated twice. Most data were analysed using an analysis of variance (ANOVA). Percentage disease incidence and foliar browning data were angularly transformed before analysis. Treatment means were compared with the least signi"cant di!erence (LSD) at a probability of 5% (P"0.05). Percentage disease incidence data from the survival experiment was expressed as mean$standard error (SE).
3. Results 3.1. Inoculum sources P. sydowiana was present on stock plants sampled from all nine C. vulgaris plant nurseries throughout the UK. The pathogen was most frequently isolated from the upper foliage and stem base of diseased stock plants (Table 1). Fewer isolates were recovered from the roots. Approximately 11}19% of soils collected from nurseries A and B were infested with P. sydowiana (Table 2). Although P. sydowiana was never isolated from new, unused propagation media, used materials were often contaminated on both nurseries. The pathogen was isolated directly from used propagation media (ca. 24}33% of samples) and media debris removed from used
Table 1 Isolation of Pestalotiopsis sydowiana from roots, stem bases/foliage of diseased stock plants of Calluna vulgaris collected from UK nurseries Plant part
Isolation (%)
Roots Stem base/foliage Both
22 41 18
Number of samples
86
Table 2 Isolation of Pestalotiopsis sydowiana from nursery materials collected from two UK nurseries producing Calluna vulgaris Isolation (%) Source
Nursery A
Nursery B
Soil Used propagation medium Used propagation trays Floor covering Dust (walkways)
19 33 25 33 32
11 24 33 20 35
(69) (30) (40) (30) (25)
Number of samples tested in parentheses.
(90) (45) (30) (44) (17)
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Table 3 Infection and disease severity of cuttings of Calluna vulgaris (cv. Kinlochruel) by Pestalotiopsis sydowiana from various sources contaminated or infected with the pathogen Source
Propagation medium Dust Cutting Propagation tray Floor covering LSD (P"0.05)
Min. period (days) after striking for infection
28 28 31 45 54 *
Disease incidence (%)
Foliar browning (%)
8 week
12 week
8 week
12 week
88 81 77 23 4
90 82 80 35 15
56 56 52 28 5
61 62 58 32 21
(71.0) (64.3) (61.7) (28.8) (11.2) (7.82)
(72.4) (65.7) (63.6) (36.0) (22.8) (7.70)
(48.5) (48.3) (45.9) (31.8) (11.8) (5.82)
(51.3) (52.0) (49.5) (34.3) (27.1) (4.90)
Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
propagation trays (ca. 25}33% of samples). P. sydowiana was also isolated from dust and soil mix samples collected from #oor covering (ca. 20}33% of samples) and walkways (ca. 32}35% of samples) within the two nurseries. Infection of cuttings of C. vulgaris (cv. Kinlochruel) by P. sydowiana was initiated from all contaminated or infected sources of the pathogen (Table 3). However, time to infection, and subsequent disease incidence and foliar browning varied considerably. Infection of cuttings was observed after 28 days when healthy cuttings were struck into uninfested propagation medium contaminated with either pathogen-infested propagation medium, or pathogen-infested dust collected from glasshouse #oors and walkways. Infection was observed after 31 days when cuttings were struck into propagation medium contaminated with a single pathogen-infected cutting, whereas it was not observed until 45 days after striking the cuttings into dirty, used trays contaminated with the pathogen. The longest time to infection occurred after 54 days when trays of healthy cuttings were placed on new, unused contaminated #oor covering. The highest disease incidence and foliar browning was observed when cuttings were struck into uninfested propagation medium contaminated with pathogen-infested propagation medium, or pathogen-infested dust collected from glasshouse #oors and walkways. Disease incidence and foliar browning of cuttings were also high when cuttings were struck into propagation medium contaminated with a single pathogen-infected cutting. The lowest levels of disease and foliar browning occurred when cuttings were struck into dirty used trays contaminated with the pathogen, and when trays of healthy cuttings were placed on unused contaminated #oor covering.
Table 4 Ability of Pestalotiopsis sydowiana-infected plant material to initiate infection of cuttings of Calluna vulgaris (cv. Bognie) following storage at 183C in the dark
3.2. Survival on infected cuttings
3.4. Ewect of tray disinfection and fungicide application
Mycelium of P. sydowiana grew within 7 days from all P. sydowiana-infected cuttings when cuttings were plated
Single and combined treatments of tray disinfection (hydrogen peroxide/peracetic acid) and a three-spray
Storage period (months)
Disease incidence (%)
0 3 6 12
57$2.6 53$3.3 49$5.4 39$4.3
Disease incidence was assessed 8 weeks after striking. Each value is the mean$standard error (SE) of "ve replicate trays, each containing 70 cuttings.
on PDAES following 0, 3, 6 and 12 months of storage at 183C in the dark. Segments of pathogen-infected cuttings sprinkled over the surface of propagation media initiated infection of cuttings (Table 4). Infection of cuttings initiated from stored pathogen-infected cuttings declined from 57% to 39% after 12 months storage. 3.3. Ewect of irrigation At the "rst disease assessment (8 weeks after striking), both disease incidence and foliar browning caused by P. sydowiana were signi"cantly greater (P"0.05) on cuttings watered from overhead compared with cuttings grown with sub-irrigation (Table 5). The disease continued to increase slightly, and at the "nal disease assessment (12 weeks after striking) both disease incidence and foliar browning were still signi"cantly greater (P"0.05) on cuttings watered from overhead.
M.P. McQuilken, K.E. Hopkins / Crop Protection 20 (2001) 591}597 Table 5 E!ect of overhead and sub-irrigation on the incidence and severity of Pestalotiopsis sydowiana on cuttings of Calluna vulgaris (cv. Alba Praecox) Disease incidence (%) Foliar browning (%) Irrigation
8 week
12 week
8 week
12 week
Overhead Sub-irrigation LSD (P"0.05)
39 (38.7) 17 (24.3) (3.33)
45 (42.3) 25 (29.6) (6.26)
32 (34.1) 16 (23.3) (6.34)
37 (37.5) 22 (27.7) (7.91)
Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
Table 6 E!ect of tray disinfection and fungicide application on the incidence and severity of Pestalotiopsis sydowiana on cuttings of Calluna vulgaris (cv. Alba Praecox) Disease incidence (%) Foliar browning (%) Treatment
8 week
12 week
8 week
12 week
Control (H O) Hydrogen peroxide/ peracetic acid Prochloraz (P) Hydrogen peroxide/ peracetic acid#P LSD (P"0.05)
39 (38.9) 17 (24.3)
46 (42.9) 20 (26.4)
32 (34.6) 11 (18.6)
38 (37.9) 13 (20.5)
7 (13.4) 2 (6.2)
10 (18.0) 6 (14.5)
2 (7.6) 1 (4.4)
8 (16.8) 4 (11.6)
(4.62)
(2.14)
(3.84)
(5.52)
Trays contaminated with P. sydowiana were dipped for 1 h; a 3-spray programme of prochloraz was applied at striking and at 14 day intervals thereafter. Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
fungicide programme of prochloraz signi"cantly reduced (P"0.05) disease incidence and foliar browning of cuttings compared to dipping trays in water (control) (Table 6). However, there were di!erences between treatments. Although the combined tray disinfection and fungicide programme signi"cantly reduced (P"0.05) disease incidence compared to tray disinfection or fungicide application alone, it did not signi"cantly reduce foliar browning. The fungicide programme of prochloraz alone was signi"cantly better (P"0.05) than dipping trays in disinfectant, and reduced both disease incidence and foliar browning.
595
4. Discussion The results from the sampling experiments have clearly identi"ed the possible sources of P. sydowiana within C. vulgaris nurseries. Primary sources of the pathogen included diseased stock plants, soil, used propagation trays and #oor covering, as well as dust collected from glasshouse walkways. Infection of cuttings by P. sydowiana was also initiated from all arti"cially contaminated or infected sources of the pathogen, although the time to infection varied considerably. The pathogen was never isolated from new materials. The presence of P. sydowiana on used propagation trays from the previous crop indicates that the pathogen can survive in deposits of dry growing media. Other pathogens of C. vulgaris, including Rhizoctonia, are reported to survive in deposits of growing media on propagation trays and polythene sheeting used in plant production (Stephens et al., 1983; Litterick et al., 1995b). Our results corroborate the importance of using pathogen-free growing media, and new or disinfected nursery materials when propagating or repotting. Growers should also try to minimise contamination of nursery materials and the propagation area with pathogen-infested soil and dust. Furthermore, attempts should be made to minimise or eliminate contact of nursery materials with non-sterile soil. The presence of P. sydowiana in samples of dust and soil mix collected from walkways within the two nurseries indicates that spread of the pathogen may occur on the shoes of nursery workers. The pathogen-infested dust could be blown by air currents, and also splashed onto sand-beds and propagation beds during irrigation. Indeed, previous studies have shown that P. sydowiana is dispersed in splashed water droplets up to distances of 1 m to infect plants (Hopkins, 1996; Hopkins and McQuilken, 1997), and this study has demonstrated that pathogen-infested dust collected from glasshouse #oors and walkways can initiate infection of healthy cuttings. The isolation of P. sydowiana from the foliage of stock plants several centimetres above soil level emphasises the danger of infected cuttings acting as potential sources of P. sydowiana infection. Damaged stock plants growing under extreme temperature and low water availability are more susceptible to infection by P. sydowiana (Hopkins and McQuilken, 2000). Consequently, avoiding stock plant damage and growing plants under extreme stress as well as the use of fungicide spray programmes are recommended to minimise infection, and to prevent stock plants acting as a source of infection during propagation. Previous trials have shown that foliar applications of prochloraz, carbendazim#prochloraz or chlorothalonil are e!ective in controlling the pathogen (Hopkins, 1996; Hopkins and McQuilken, 1997; McQuilken et al., 1999).
596
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Mycelium of P. sydowiana grew from all pathogeninfected cuttings when plated onto PDAES following storage up to a year. Furthermore, debris of these stored cuttings sprinkled over propagation media initiated new infections of cuttings by the pathogen. These observations reveal the ability of the P. sydowiana to survive long term on infected plant debris, and for the debris to act as a potent source of infection. Removal of plant debris from propagation beds and subsequent disinfection at the end of propagating should help to prevent debris acting as a source of infection before a new crop is established. Irrigation method was shown to a!ect the incidence and severity of P. sydowiana on cuttings. Disease was less on cuttings watered by sub-irrigation compared with watering from overhead. Previous trials have also revealed that other foliar pathogens of C. vulgaris, including Cylindrocarpon and Botrytis cinerea, are encouraged by overhead irrigation (O'Neill and McQuilken, 2000; McQuilken, 2001). Adoption of sub-irrigation rather than overhead would appear to be a useful component for integrated control of P. sydowiana as well as other foliar pathogens a!ecting C. vulgaris. However, sub-irrigation alone is unlikely to provide commercially acceptable disease control. Future trials are planned to investigate integration of foliar-applied fungicides with irrigation method. The tray disinfection and fungicide trial demonstrated, for the "rst time, the bene"ts of combining disinfection and fungicide treatments. Although tray disinfection with hydrogen peroxide/peracetic acid alone reduced the disease considerably, it is most unlikely to provide complete control where there is a high inoculum of the pathogen. The combined tray disinfection and fungicide treatment reduced disease incidence compared to tray disinfection or fungicide application alone. Consequently, growers should consider supplementing disinfection with foliar applications of fungicide when reusing propagation trays and if inoculum levels of P. sydowiana are high. The activity of disinfectants against a wide range of plant pathogens is well established, and they have been shown to be e!ective in disinfecting a wide range of nursery materials other than propagation trays, including capillary matting, #oor coverings as well as irrigation water and lines (Pilgaard, 1990; Loschenkohl et al., 1990; Lin"eld, 1991; O'Neill, 1995, 1997; Hingley and Pearce, 1998). They should be used as part of a comprehensive hygiene programme to prevent introduction of pathogens, especially during propagation. In conclusion, it is clear that simple hygiene measures in combination with good crop management practices and wise fungicide application are likely to be most important in future integrated control of P. sydowiana on ericaceous plants.
Acknowledgements The authors thank all the UK growers for providing samples of diseased plants and allowing sampling to be conducted on their nurseries. We also thank Mr. Nigel Willis of Highland Heathers Ltd., Argyll, Scotland for providing healthy cuttings for use in trials, Jim Thomson for technical assistance and Lynda Raynor for help with the statistical analyses. Financial support for this work from growers through the Horticultural Development Council (HDC) is gratefully acknowledged. The Scottish Agricultural College receives support from the Scottish Executive Rural A!airs Department (SERAD).
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Sources, survival and management of Pestalotiopsis sydowiana on Calluna vulgaris nurseries M.P. McQuilken*, K.E. Hopkins Department of Plant Biology, The Scottish Agricultural College, Auchincruive, Ayr, KA6 5HW, UK Received 9 October 2000; received in revised form 12 January 2001; accepted 12 January 2001
Abstract Sources of Pestalotiopsis sydowiana were determined on commercial nurseries producing Calluna vulgaris throughout the UK. The pathogen was isolated from diseased stock plants, nursery soils, used growing media, propagation trays and #oor covering and dust collected from greenhouse walkways. P. sydowiana was not isolated from new or unused materials. Infection of cuttings of C. vulgaris was initiated from a range of contaminated or infected sources. The pathogen was able to survive on infected plant debris for up to 1 year to initiate new infections. Greenhouse trials were conducted to evaluate the e!ects of disease management methods (irrigation, tray disinfection and fungicide application) on control during the propagation of C. vulgaris. Disease incidence and foliar browning caused by P. sydowiana were less on cuttings watered by sub-irrigation compared with watering from overhead. Single and combined treatments of tray disinfection (hydrogen peroxide/peracetic acid) and a three-spray programme of prochloraz to the foliage of cuttings signi"cantly reduced disease incidence and foliar browning compared to dipping trays in water. The combined tray disinfection and fungicide programme signi"cantly reduced disease incidence but not foliar browning, compared to tray disinfection or fungicide application alone. Fungicide application alone was better than disinfection. The importance of these "ndings for the integrated control of P. sydowiana on ericaceous plant nurseries is discussed. 2001 Elsevier Science Ltd. All rights reserved. Keywords: Pestalotiopsis sydowiana; Calluna vulgaris; Inoculum sources; Survival; Irrigation; Disinfectants; Fungicides
1. Introduction Previous studies on the biology and control of diseases in crops of Calluna vulgaris (L.) Hull and other ericaceous crops have focused on the pathogens Rhizoctonia, Phytophthora and Pythium spp. (Rutherford et al., 1989; Backhaus and Leopold, 1990; Harig and Backhaus, 1990; Litterick and Holmes, 1990; Litterick et al., 1995a, b; Litterick and McQuilken, 1998). Advances in the technology of production systems and increased knowledge of pathogen biology have led to improved control of these pathogens. However, growers of C. vulgaris continue to report heavy crop losses due to root and foliar diseases. During recent seasons, an increasing number of damaging infections of C. vulgaris, caused by Pestalotiopsis spp., have been found on plant samples submitted by growers
* Corresponding author. Fax: #44-1292-525314. E-mail address: [email protected] (M.P. McQuilken). Present address: Crop Protection Compendium, CAB International, Wallingford OX10 8DE, UK.
to the SAC Crop Health Centre. Serious damage has been seen on cuttings, potted-on-plants and stock plants. The symptoms are a necrosis and death of the foliage, with extensive acervuli production on a!ected plant parts (Hopkins, 1996). Sometimes, a general rotting of the root and stem base may also occur (Hopkins, 1996; Hopkins and McQuilken, 1997, 2000). Recent studies have shown that P. sydowiana is the predominant species of Pestalotiopsis associated with C. vulgaris as well as other ericaceous plants in the UK (Hopkins and McQuilken, 2000). Pathogenicity tests and host range studies have also indicated that isolates of P. sydowiana are not host speci"c and they can infect a wide range of hardy ornamental species (Hopkins and McQuilken, 2000). In view of the exchange of ericaceous plants and other hardy ornamentals between the UK and other European countries, it is likely that P. sydowiana may have become established throughout most Northern European countries. It is clear that P. sydowiana poses a real threat to the production of C. vulgaris by causing plant losses, reduced plant quality and disruption of production schedules;
0261-2194/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 1 - 2 1 9 4 ( 0 1 ) 0 0 0 2 8 - X
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severe disease symptoms often cause the plants to be unsaleable (Hopkins, 1996; McQuilken et al., 1999). Extensive use of polythene tunnels, fertilizers, controlled irrigation and large-scale monoculture of C. vulgaris, particularly in Scotland has created ideal conditions for the development of the disease (Hopkins and McQuilken, 1997). As P. sydowiana is di$cult to eliminate completely once it is introduced into nurseries, disease control may be more e!ective if based on a knowledge of the potential sources and survival of the pathogen. However, at present, there is very little such information available on P. sydowiana. The objectives of this study were to (i) identify sources of initial inoculum of P. sydowiana on nurseries producing C. vulgaris, and to assess whether infection by the pathogen could be initiated from such inoculum sources; (ii) monitor the survival of the pathogen on infected plant material; (iii) evaluate the e!ects of various disease management methods (irrigation, tray disinfection and fungicide application) on control. A brief preliminary report has already been published (McQuilken et al., 1999).
2. Materials and methods 2.1. Identixcation of inoculum sources To determine if stock plants of C. vulgaris were infected with P. sydowiana, samples with foliar browning, root and stem-base rots were collected from several nurseries in the UK. Diseased plant tissue (3}5 mm was surface sterilised in 10% (v/v) sodium hypochlorite for 5}10 s and rinsed in three changes of sterile distilled water. Surfacesterilised segments of tissue were blot-dried on sterile "lter paper and plated on Oxoid potato dextrose agar (PDA; Unipath, Basingstoke, Hants., UK) modi"ed to contain 100 g ml\ of erythromycin and streptomycin (PDAES). Plates were incubated at 203C, and after 3}5 days, mycelia typical of P. sydowiana were transferred to single 9 cm diameter Petri dishes of PDAES. Identi"cation of isolates of P. sydowiana was made based on morphological and cultural characteristics (Sutton, 1980). Samples of soil, used ericaceous plant growing medium, growing medium debris removed from used propagation trays and #oor covering, and dust swept from glasshouse walkways from two nurseries were tested for the presence of P. sydowiana. Samples were air-dried, sieved through a 2 mm sieve and 10 cm of each was shaken in 90 ml of sterile 0.01% agar on a wrist action shaker at low speed for 10 min. After standing for a further 20 min, a dilution series was made and 0.5 ml samples of the dilutions were spread-plated onto PDAES containing Triton X-100 (2 ml l\; BDH Laboratory Supplies, Poole, UK). Colonies of P. sydowiana were identi"ed after 7}10 days growth at 203C.
A greenhouse trial was conducted to assess whether infection of cuttings of C. vulgaris (cv. Kinlochruel) by P. sydowiana could be initiated from a range of potential inoculum sources. Cuttings obtained from disease-free 3-year-old stock plants were struck (70 per tray) into propagation medium (80 : 20 "ne peat : super "ne perlite; Bulrush Peat Company Ltd., Bellagly, N. Ireland) in 70-cell plastic plug trays (28;16 cm with 70;10 ml individual plugs). These trays were used in all subsequent trials. Preliminary trials indicated that prior wounding of plant tissue by dusting lightly with carborundum was necessary to establish infection of cuttings (Hopkins and McQuilken, 2000). Treatments included: (1) the incorporation of pathogen-infested propagation medium into trays of uninfested medium (1:10 v/v); (2) sprinkling samples (approximately 3 g per replicate tray) of pathogeninfested dust, collected from glasshouse #oors and walkways, over trays immediately before striking; (3) striking two pathogen-infected cuttings into the trays (two centre rows) of healthy cuttings; (4) dirty, used trays contaminated with the pathogen; (5) new, unused trays placed on Mypex (LBS Group, Colne, Lancs., UK) #oor covering contaminated with the pathogen. Appropriate control treatments were also set up using uncontaminated materials. Trays were placed under low polythene tunnels within a greenhouse maintained at 12}243C, and watered weekly by overhead irrigation. After 8 and 12 weeks, individual cuttings were assessed for incidence of Pestalotiopsis (presence of black spore masses) and foliar browning using a percentage browning scale of 0}100, where 0"no foliar browning and 100"foliage totally brown. To con"rm infection by P. sydowiana after 12 weeks, small pieces of diseased tissue were plated on PDAES and observed for colonies typical of the pathogen. 2.2. Survival on infected cuttings Shoots of cuttings infected with P. sydowiana were air-dried and stored in sealed plastic boxes (16;28;5 cm) at 183C in the dark. The ability of P. sydowiana to grow from stored cuttings was assessed after 0, 3, 6 and 12 months by plating 3}5 mm segments (4 per dish) of four cuttings on PDAES in 9 cm diameter Petri dishes. After 7}10 days of incubation at 203C, the number of cuttings giving rise to P. sydowiana was counted. The ability of P. sydowiana-infected plant material (cuttings) to initiate infection after each storage period was also evaluated by conducting a small-scale glasshouse trial. Four cuttings were removed from storage, cut into segments and sprinkled over the surface of "ve replicate plug trays containing uninfested propagation medium, prior to striking 70 pathogen-free cuttings (cv. Bognie) per tray. Trays of cuttings were wounded, placed in a glasshouse under low polythene tunnels, watered,
M.P. McQuilken, K.E. Hopkins / Crop Protection 20 (2001) 591}597
and assessed for disease incidence and severity as described before. Controls consisted of pathogen-free cuttings struck into uninfested propagation medium. 2.3. Ewect of irrigation A greenhouse trial was conducted to investigate the e!ect of di!erent irrigation methods on the incidence and severity of Pestalotiopsis. Cuttings (cv. Alba Praecox) were struck into plug trays as described before, except that two of the cuttings in the central two rows of each tray were infected with P. sydowiana as inoculum. Trays were placed on capillary sand-beds (for sub-irrigation) or Mypex (for weekly overhead watering) under low polythene tunnels within the glasshouse. Trays of cuttings were wounded, and assessed subsequently for disease incidence and severity as described before. 2.4. Ewect of tray disinfection and fungicide application A greenhouse trial was conducted to evaluate tray disinfection alone and in combination with a fungicide applied to cuttings on the incidence and severity of Pestalotiopsis. The fungicide, prochloraz, was selected as it is recommended for control of Pestalotiopsis on ericaceous plants, and gave good control against the pathogen on C. vulgaris in previous trials (McQuilken et al., 1997). To obtain trays contaminated with P. sydowiana, wounded cuttings were grown in new, unused trays and arti"cially inoculated by brushing individual cuttings with a spore suspension (1}3;10 conidia ml\). The spore suspension was prepared by #ooding 21-day-old PDA Petri dish cultures with sterile distilled water and gently scraping the colony surface with a sterile bent glass rod. After 12 weeks, infected cuttings were removed and contaminated trays were used in the trial. There were "ve treatments: (1) control, trays dipped in water for 1 h; (2) trays dipped in hydrogen peroxide (H O )#peracetic acid (CH CO.OOH) (Jet 5, 23%/5% s.c., Hortichem Ltd, Amesbury, Wilts., UK) for 1 h; (3) a three-spray programme of prochloraz (Octave, 46% w/w w.p., Scotts UK Professional, Ipswich, UK) at 0.46 g a.i. l\ applied at striking and at 14 day intervals thereafter; (4) combined treatments of 2 and 3; and (5) new, unused trays dipped in water for 1 h. The recommended rate of Jet 5 in water was used (1 : 125 v/v), and loose growing media and crop debris were removed by shaking prior to dipping. Following treatment, all trays were rinsed in water, allowed to dry, re"lled with propagation media and restruck with cuttings (cv. Alba Praecox). The fungicide sprays were applied to cuttings to run o!, using a Mardrive Precision sprayer at 300 kPa. Trays of cuttings were wounded, and assessed subsequently for disease incidence and severity as described before.
593
2.5. Statistical analyses All glasshouse trials were arranged in randomised complete block designs with "ve replicates for each treatment, and repeated twice. Most data were analysed using an analysis of variance (ANOVA). Percentage disease incidence and foliar browning data were angularly transformed before analysis. Treatment means were compared with the least signi"cant di!erence (LSD) at a probability of 5% (P"0.05). Percentage disease incidence data from the survival experiment was expressed as mean$standard error (SE).
3. Results 3.1. Inoculum sources P. sydowiana was present on stock plants sampled from all nine C. vulgaris plant nurseries throughout the UK. The pathogen was most frequently isolated from the upper foliage and stem base of diseased stock plants (Table 1). Fewer isolates were recovered from the roots. Approximately 11}19% of soils collected from nurseries A and B were infested with P. sydowiana (Table 2). Although P. sydowiana was never isolated from new, unused propagation media, used materials were often contaminated on both nurseries. The pathogen was isolated directly from used propagation media (ca. 24}33% of samples) and media debris removed from used
Table 1 Isolation of Pestalotiopsis sydowiana from roots, stem bases/foliage of diseased stock plants of Calluna vulgaris collected from UK nurseries Plant part
Isolation (%)
Roots Stem base/foliage Both
22 41 18
Number of samples
86
Table 2 Isolation of Pestalotiopsis sydowiana from nursery materials collected from two UK nurseries producing Calluna vulgaris Isolation (%) Source
Nursery A
Nursery B
Soil Used propagation medium Used propagation trays Floor covering Dust (walkways)
19 33 25 33 32
11 24 33 20 35
(69) (30) (40) (30) (25)
Number of samples tested in parentheses.
(90) (45) (30) (44) (17)
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Table 3 Infection and disease severity of cuttings of Calluna vulgaris (cv. Kinlochruel) by Pestalotiopsis sydowiana from various sources contaminated or infected with the pathogen Source
Propagation medium Dust Cutting Propagation tray Floor covering LSD (P"0.05)
Min. period (days) after striking for infection
28 28 31 45 54 *
Disease incidence (%)
Foliar browning (%)
8 week
12 week
8 week
12 week
88 81 77 23 4
90 82 80 35 15
56 56 52 28 5
61 62 58 32 21
(71.0) (64.3) (61.7) (28.8) (11.2) (7.82)
(72.4) (65.7) (63.6) (36.0) (22.8) (7.70)
(48.5) (48.3) (45.9) (31.8) (11.8) (5.82)
(51.3) (52.0) (49.5) (34.3) (27.1) (4.90)
Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
propagation trays (ca. 25}33% of samples). P. sydowiana was also isolated from dust and soil mix samples collected from #oor covering (ca. 20}33% of samples) and walkways (ca. 32}35% of samples) within the two nurseries. Infection of cuttings of C. vulgaris (cv. Kinlochruel) by P. sydowiana was initiated from all contaminated or infected sources of the pathogen (Table 3). However, time to infection, and subsequent disease incidence and foliar browning varied considerably. Infection of cuttings was observed after 28 days when healthy cuttings were struck into uninfested propagation medium contaminated with either pathogen-infested propagation medium, or pathogen-infested dust collected from glasshouse #oors and walkways. Infection was observed after 31 days when cuttings were struck into propagation medium contaminated with a single pathogen-infected cutting, whereas it was not observed until 45 days after striking the cuttings into dirty, used trays contaminated with the pathogen. The longest time to infection occurred after 54 days when trays of healthy cuttings were placed on new, unused contaminated #oor covering. The highest disease incidence and foliar browning was observed when cuttings were struck into uninfested propagation medium contaminated with pathogen-infested propagation medium, or pathogen-infested dust collected from glasshouse #oors and walkways. Disease incidence and foliar browning of cuttings were also high when cuttings were struck into propagation medium contaminated with a single pathogen-infected cutting. The lowest levels of disease and foliar browning occurred when cuttings were struck into dirty used trays contaminated with the pathogen, and when trays of healthy cuttings were placed on unused contaminated #oor covering.
Table 4 Ability of Pestalotiopsis sydowiana-infected plant material to initiate infection of cuttings of Calluna vulgaris (cv. Bognie) following storage at 183C in the dark
3.2. Survival on infected cuttings
3.4. Ewect of tray disinfection and fungicide application
Mycelium of P. sydowiana grew within 7 days from all P. sydowiana-infected cuttings when cuttings were plated
Single and combined treatments of tray disinfection (hydrogen peroxide/peracetic acid) and a three-spray
Storage period (months)
Disease incidence (%)
0 3 6 12
57$2.6 53$3.3 49$5.4 39$4.3
Disease incidence was assessed 8 weeks after striking. Each value is the mean$standard error (SE) of "ve replicate trays, each containing 70 cuttings.
on PDAES following 0, 3, 6 and 12 months of storage at 183C in the dark. Segments of pathogen-infected cuttings sprinkled over the surface of propagation media initiated infection of cuttings (Table 4). Infection of cuttings initiated from stored pathogen-infected cuttings declined from 57% to 39% after 12 months storage. 3.3. Ewect of irrigation At the "rst disease assessment (8 weeks after striking), both disease incidence and foliar browning caused by P. sydowiana were signi"cantly greater (P"0.05) on cuttings watered from overhead compared with cuttings grown with sub-irrigation (Table 5). The disease continued to increase slightly, and at the "nal disease assessment (12 weeks after striking) both disease incidence and foliar browning were still signi"cantly greater (P"0.05) on cuttings watered from overhead.
M.P. McQuilken, K.E. Hopkins / Crop Protection 20 (2001) 591}597 Table 5 E!ect of overhead and sub-irrigation on the incidence and severity of Pestalotiopsis sydowiana on cuttings of Calluna vulgaris (cv. Alba Praecox) Disease incidence (%) Foliar browning (%) Irrigation
8 week
12 week
8 week
12 week
Overhead Sub-irrigation LSD (P"0.05)
39 (38.7) 17 (24.3) (3.33)
45 (42.3) 25 (29.6) (6.26)
32 (34.1) 16 (23.3) (6.34)
37 (37.5) 22 (27.7) (7.91)
Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
Table 6 E!ect of tray disinfection and fungicide application on the incidence and severity of Pestalotiopsis sydowiana on cuttings of Calluna vulgaris (cv. Alba Praecox) Disease incidence (%) Foliar browning (%) Treatment
8 week
12 week
8 week
12 week
Control (H O) Hydrogen peroxide/ peracetic acid Prochloraz (P) Hydrogen peroxide/ peracetic acid#P LSD (P"0.05)
39 (38.9) 17 (24.3)
46 (42.9) 20 (26.4)
32 (34.6) 11 (18.6)
38 (37.9) 13 (20.5)
7 (13.4) 2 (6.2)
10 (18.0) 6 (14.5)
2 (7.6) 1 (4.4)
8 (16.8) 4 (11.6)
(4.62)
(2.14)
(3.84)
(5.52)
Trays contaminated with P. sydowiana were dipped for 1 h; a 3-spray programme of prochloraz was applied at striking and at 14 day intervals thereafter. Disease incidence and foliar browning were assessed 8 and 12 weeks after striking. Values in parentheses are angular transformations of percentage means from "ve replicate trays, each containing 70 cuttings. LSD is the least signi"cant di!erence at a probability of 5% (P"0.05).
fungicide programme of prochloraz signi"cantly reduced (P"0.05) disease incidence and foliar browning of cuttings compared to dipping trays in water (control) (Table 6). However, there were di!erences between treatments. Although the combined tray disinfection and fungicide programme signi"cantly reduced (P"0.05) disease incidence compared to tray disinfection or fungicide application alone, it did not signi"cantly reduce foliar browning. The fungicide programme of prochloraz alone was signi"cantly better (P"0.05) than dipping trays in disinfectant, and reduced both disease incidence and foliar browning.
595
4. Discussion The results from the sampling experiments have clearly identi"ed the possible sources of P. sydowiana within C. vulgaris nurseries. Primary sources of the pathogen included diseased stock plants, soil, used propagation trays and #oor covering, as well as dust collected from glasshouse walkways. Infection of cuttings by P. sydowiana was also initiated from all arti"cially contaminated or infected sources of the pathogen, although the time to infection varied considerably. The pathogen was never isolated from new materials. The presence of P. sydowiana on used propagation trays from the previous crop indicates that the pathogen can survive in deposits of dry growing media. Other pathogens of C. vulgaris, including Rhizoctonia, are reported to survive in deposits of growing media on propagation trays and polythene sheeting used in plant production (Stephens et al., 1983; Litterick et al., 1995b). Our results corroborate the importance of using pathogen-free growing media, and new or disinfected nursery materials when propagating or repotting. Growers should also try to minimise contamination of nursery materials and the propagation area with pathogen-infested soil and dust. Furthermore, attempts should be made to minimise or eliminate contact of nursery materials with non-sterile soil. The presence of P. sydowiana in samples of dust and soil mix collected from walkways within the two nurseries indicates that spread of the pathogen may occur on the shoes of nursery workers. The pathogen-infested dust could be blown by air currents, and also splashed onto sand-beds and propagation beds during irrigation. Indeed, previous studies have shown that P. sydowiana is dispersed in splashed water droplets up to distances of 1 m to infect plants (Hopkins, 1996; Hopkins and McQuilken, 1997), and this study has demonstrated that pathogen-infested dust collected from glasshouse #oors and walkways can initiate infection of healthy cuttings. The isolation of P. sydowiana from the foliage of stock plants several centimetres above soil level emphasises the danger of infected cuttings acting as potential sources of P. sydowiana infection. Damaged stock plants growing under extreme temperature and low water availability are more susceptible to infection by P. sydowiana (Hopkins and McQuilken, 2000). Consequently, avoiding stock plant damage and growing plants under extreme stress as well as the use of fungicide spray programmes are recommended to minimise infection, and to prevent stock plants acting as a source of infection during propagation. Previous trials have shown that foliar applications of prochloraz, carbendazim#prochloraz or chlorothalonil are e!ective in controlling the pathogen (Hopkins, 1996; Hopkins and McQuilken, 1997; McQuilken et al., 1999).
596
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Mycelium of P. sydowiana grew from all pathogeninfected cuttings when plated onto PDAES following storage up to a year. Furthermore, debris of these stored cuttings sprinkled over propagation media initiated new infections of cuttings by the pathogen. These observations reveal the ability of the P. sydowiana to survive long term on infected plant debris, and for the debris to act as a potent source of infection. Removal of plant debris from propagation beds and subsequent disinfection at the end of propagating should help to prevent debris acting as a source of infection before a new crop is established. Irrigation method was shown to a!ect the incidence and severity of P. sydowiana on cuttings. Disease was less on cuttings watered by sub-irrigation compared with watering from overhead. Previous trials have also revealed that other foliar pathogens of C. vulgaris, including Cylindrocarpon and Botrytis cinerea, are encouraged by overhead irrigation (O'Neill and McQuilken, 2000; McQuilken, 2001). Adoption of sub-irrigation rather than overhead would appear to be a useful component for integrated control of P. sydowiana as well as other foliar pathogens a!ecting C. vulgaris. However, sub-irrigation alone is unlikely to provide commercially acceptable disease control. Future trials are planned to investigate integration of foliar-applied fungicides with irrigation method. The tray disinfection and fungicide trial demonstrated, for the "rst time, the bene"ts of combining disinfection and fungicide treatments. Although tray disinfection with hydrogen peroxide/peracetic acid alone reduced the disease considerably, it is most unlikely to provide complete control where there is a high inoculum of the pathogen. The combined tray disinfection and fungicide treatment reduced disease incidence compared to tray disinfection or fungicide application alone. Consequently, growers should consider supplementing disinfection with foliar applications of fungicide when reusing propagation trays and if inoculum levels of P. sydowiana are high. The activity of disinfectants against a wide range of plant pathogens is well established, and they have been shown to be e!ective in disinfecting a wide range of nursery materials other than propagation trays, including capillary matting, #oor coverings as well as irrigation water and lines (Pilgaard, 1990; Loschenkohl et al., 1990; Lin"eld, 1991; O'Neill, 1995, 1997; Hingley and Pearce, 1998). They should be used as part of a comprehensive hygiene programme to prevent introduction of pathogens, especially during propagation. In conclusion, it is clear that simple hygiene measures in combination with good crop management practices and wise fungicide application are likely to be most important in future integrated control of P. sydowiana on ericaceous plants.
Acknowledgements The authors thank all the UK growers for providing samples of diseased plants and allowing sampling to be conducted on their nurseries. We also thank Mr. Nigel Willis of Highland Heathers Ltd., Argyll, Scotland for providing healthy cuttings for use in trials, Jim Thomson for technical assistance and Lynda Raynor for help with the statistical analyses. Financial support for this work from growers through the Horticultural Development Council (HDC) is gratefully acknowledged. The Scottish Agricultural College receives support from the Scottish Executive Rural A!airs Department (SERAD).
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