‡Permanent address: Faculty of Science, University of Ain-Shams, Department of Microbiology, Cairo, 11566, Egypt.
A highly virulent and polyvalent Streptomyces phage was isolated from a potato field near Albany, Western Australia. The efficacy of the isolated phage to disinfest seed potato tubers artificially inoculated with a common scab-causing streptomycete was evaluated. The phage suspension was prepared in a mini-bioreactor. Diseased potatoes were bathed in a phage suspension (1 × 109 plaque-forming units per mL) for 24 h. The suspension was constantly circulated within a novel 25 L phage bath by means of an air-sparging pipe driven from an air compressor. Phage-treated scab-affected seed potatoes planted into free-draining polystyrene boxes containing steam-pasteurized field soil produced tuber progeny with significantly (P < 0·05) reduced levels of surface lesions of scab (1·2%) compared with tubers harvested from nonphage-treated tubers (23%). The number of scab lesions was also significantly reduced (P < 0·05) by phage treatment of mother tubers. No significant differences were recorded in weight, size or number of harvested tubers from phage-treated or nontreated mother tubers. This is the first in vivo study that has used Streptomyces phage to significantly disinfest seed potatoes of Streptomyces scabies and thereby reduce contamination of soil from seed-tuber-borne inoculum and reduce infection of daughter tubers.
In Australia, potatoes (Solanum tuberosum) are of significant horticultural importance. A substantial domestic market exists alongside a well developed and increasing export marketplace from Australia. Crops are cultivated once or twice a year depending on the area (T. Carstairs, Potato Growers Association, Western Australia, personal communication, 1998). Intensive and repeated cropping may have contributed to the increased incidence of common scab caused by Streptomyces scabies, which now represents a serious problem for potato growers.
Common scab disease in potato crops causes significant income losses to growers throughout the world. Losses are primarily due to grade reductions of crops as a result of diminished cosmetic appearance, but acceptance in the marketplace of infected crops grown as seed tubers is also lowered (Hooker, 1981). The disease is characterized by deep or superficial corky lesions that are incited by streptomycetes historically referred to as S. scabies. However, a number of other morphologically unrelated species of streptomycetes, with varying levels of DNA relatedness (Faucher et al., 1995) have been described that cause identical symptoms (Doering-Saad et al., 1992). The disease is caused by infection of expanding epidermal lenticels by the pathogenic streptomycetes. These appear to be both soil- and tuber-borne, and in general, scab severity increases linearly with increasing soil inoculum (Keinath & Loria, 1995).
Seed disinfestation treatments have delivered variable outcomes. Some have met with success whilst others have not or are no longer available. These include preplanting treatment of tubers with boric acid solution (1–3%), which provided significant disease control (De & Sengupta, 1992), and preplanting tuber dusting with pentachloronitrobenzene (PCNB), which offered some level of protection (Hooker, 1981), but concerns with carcinogenic breakdown products have limited current use (Locci, 1994).
Previous studies have included the search for rare nonstreptomycete actinomycetes for a diverse array of purposes (El-Tarabily et al., 2000). Many of these rare species are slow-growing and may be completely overgrown by more rapidly growing species. Many fast-growing species, including streptomycetes, are routinely eliminated from the growing surface of a laboratory plate by inoculation with phages active against these hosts following the method described by El-Tarabily et al. (2000). It was therefore postulated that the effect could be replicated in vivo by treating contaminated tubers in the same way with some modifications to phage production that would allow the preparation of phages on a commercial scale.
The aim of this study was to develop and evaluate a rapid technique to propagate large volumes of pathogen and phage with an inexpensive air-lift-type mini-bioreactor and to evaluate further the impact and effect of disinfesting scab-infected seed potatoes with a highly virulent and polyvalent Streptomyces phage to minimize or eliminate the risk of contaminating noninfested soil by way of seed-tuber-borne pathogen inoculum.
Materials and methods
Diseased tuber and soil collection
Soil and commercially grown, naturally infected potatoes (cv. Kennebec) exhibiting common scab lesions were collected from a commercial potato farm near Albany, 400 km south of Perth, Western Australia. Percentage surface area affected by lesions on these tubers ranged from 10 to 40%. Most lesions were discrete, but some had coalesced into large irregular shapes covering up to 40% of the tuber surface. Lesions exhibited a raised corky appearance. No deep-pitted lesions were evident. The potatoes were used for pathogen isolation.
The soil was well drained and no compaction layer was observed at a depth of 0·5 m. Soil pH was 7·2 (0·01 m CaCl2). Samples were collected randomly from two adjacent fields (five soil samples and 10 infected tubers per field). Soil samples were collected up to a depth of ≈ 20 cm and mixed to ensure uniformity. Bulked soil was passed through a 3-mm sieve to remove stones and root fragments, and used for phage isolation.
Isolation of streptomycetes associated with common scab lesions on potato
Isolation of streptomycetes from scab lesions followed the method as described by Harrison (1962). Surface-sterilized potato skin segments were sliced into ≈ 1-mm-thick sections and placed onto starch-casein agar plates (Küster & Williams, 1964). Nystatin and cycloheximide (50 μg mL−1) were added to the cooled (45°C) sterile molten agar immediately prior to pouring plates to reduce fungal growth (Williams & Davies, 1965). Poured plates were dried in a laminar-flow cabinet for 30 min. After 7 days of incubation at 28°C (± 2°C), colonies exhibiting aerial hyphae with Streptomyces morphology were isolated and streaked onto oatmeal yeast extract agar (OMY). Cultures were incubated in the dark at 28°C (± 2°C) for 14 days. In all subsequent experiments, the incubation temperature was 28°C (± 2°C) for Streptomyces spp. and phage propagation unless otherwise stated.
As a preliminary criterion for further study, streptomycetes grown on OMY and exhibiting grey, spiral spore chains were transferred to peptone-yeast extract-iron agar (Tresner & Danga, 1958) and tyrosine agar (Shirling & Gottlieb, 1966) plates. The production of melanoid pigments characteristic of scab-causing S. scabies was visible after 4 days' incubation in the dark, resulting in one isolate, S. scabies AS17, being selected for subsequent experiments.
Preparation of pathogen inoculum
Inoculum was prepared by scraping 20 plates of sporulating cultures, grown on OMY, into 1 L of sterile distilled water, and 200 mL of this suspension was added to 10 L of sterilized (121°C for 20 min) fishmeal broth (El-Tarabily et al., 2000) that had been aseptically introduced into a mini-bioreactor (Fig. 1) and incubated for 10 days. Inoculum was used immediately. Serial dilutions were prepared and five 0·2 mL aliquots were inoculated separately onto OMY plates before use to confirm the presence or absence of isolate AS17 and inoculum density.
Evaluation of pathogenicity and production of diseased seed tubers
Parental generation mini-tubers (cv. Kennebec) (Agriculture Victoria, Toolangi, Victoria, Australia) were surface-sterilized by immersion in 0·6% NaOCl for 10 min, rinsed three times in distilled water and left to dry. Dried mini-tubers were soaked in the prepared pathogen inoculum (1 × 1010 cfu mL−1) for 1 h, removed wet and immediately planted into free-draining polystyrene boxes (450 × 450 × 400 mm) containing potato field soil from Albany. Soil had been steam-pasteurized for 1 h at 60°C on three consecutive days and was moist at planting. Six mini-tubers were planted per box at a depth of ≈ 5 cm. A further 40 mL of pathogen inoculum was poured around each tuber. Containers were watered daily for the first 2 weeks and sparingly thereafter, as reduced water is known to promote the incidence of scab symptoms (Loria & Lambert, 1986). Liquid fertilizer (Aquasol/Hortico Aust. Pty Ltd, Victoria, Australia) was applied weekly at the manufacturer's recommended rate. The treatment was replicated 10 times with six plants per box in fully randomized blocks and maintained in a glasshouse at 22°C (± 2°C). Plants were grown for 60 days. At harvest, attached tubers were removed, washed and inspected for scab infection. Disease symptoms and incidence satisfied Koch's postulates and generated a sufficient number of infected tubers for the phage experiment.
Identification of the pathogenic Streptomyces isolate
The morphological and cultural characteristics, utilization of carbon sources, pH sensitivity, resistance to antibiotics and other inhibitory compounds, as well as the degradation of complex compounds, for isolate AS17 were determined as described by Lambert & Loria (1989).
Phage isolation, purification and characterization
Streptomyces scabies AS17 was used as the propagation host for phage isolation. Erlenmyer flasks (250 mL) containing 20 mL of sterile peptone-yeast extract-calcium nitrate (PYCa) broth (Bradley et al., 1961) were inoculated with 1 mL of the spore suspension of the propagation host and 5 g of bulked soil sample and incubated in a gyratory shaker (Model G76, New Brunswick Scientific-Edison, NJ, USA) at 150 rpm for 48 h. After incubation, the suspensions from each flask were centrifuged for 1 h at 480 g and the supernatant filtered through sterile 0·22 µm membrane filters (Pall Australia, Melbourne, Victoria, Australia) and collected in sterile tubes. An aliquot (0·2 mL) of a glycerol suspension of the host was inoculated separately onto PYCa plates (five replicates) and dried for 30 min in a laminar-flow cabinet (Vickers & Williams, 1987). After drying, 0·2 mL of the soil filtrate was inoculated as a centrally placed droplet and spread over the medium surface with a sterile glass spreader (Bradley et al., 1961). The plates were incubated in the dark for 48 h and examined for lytic zones (plaques) (Williams et al., 1980). After incubation, a single plaque was aseptically removed with a scalpel and resuspended in 100 mL of PYCa broth inoculated with 5 mL of broth culture of S. scabies AS17 and incubated for 48 h. The broth was filtered through Whatman paper no. 1 (Whatman International Ltd, Maidstone, UK) and filtered again through sterile 0·22 µm membrane filters. The procedure was repeated until a high titre of 1 × 1012 plaque-forming units (PFU) mL−1 of phage suspension was obtained. The purified phage (ØAS1) suspension was stored at 4°C (Williams et al., 1980).
The morphology and diameter of single plaques were examined by inoculating PYCa agar with a mixture of 0·2 mL of the host (1 × 1010 cfu mL−1) and the same volume of ØAS1 suspension (1 × 1012 PFU mL−1) and incubating for 48 h.
Morphology of the phage ØAS1 was examined by transmission electron microscope (JEOL-2000 FX II) operating at 80 kV at a magnification of ×250 000. A drop of phage suspension was placed on 200-gauge copper grids with carbon-coated formvar films and the excess drawn off with filter paper. A saturated solution of uranyl acetate was placed on the grids and the excess drawn off as before.
The polyvalency of ØAS1 was determined following the method described by El-Tarabily et al. (1995). Lytic zones were recorded 4 days later.
Mini-bioreactor design and construction
To propagate enough ØAS1 to complete the disinfestation of a large number of tubers, a volume of ≈ 40 L (1 × 109 PFU mL−1) of phage was required. An inexpensive and simple air-lift mini-bioreactor was designed and constructed (Fig. 1). The outside wall of the bioreactor was constructed from white PVC tubing (4 mm wall thickness) with a threaded section attached at the upper end. The overall dimensions of this section were 800 mm long and 150 mm in diameter. The lower end was fitted with a conical PVC reducing section leading to a tap and drain. The tap and drain were used to empty the contents of the mini-bioreactor and also used as the point of injection of sterilizing gasses. The top was fitted with a PVC screw cap fitted with an internal ‘O’ ring seal. A clear acrylic inspection window (100 mm in diameter) was attached over a centrally cut-out section (80 mm in diameter) on top of the lid and sealed with a silicone sealant. A screw tube (10 mm in diameter) was sealed into the main reactor wall 30 mm below the lower edge of the screw cap. Diagonally opposite, a second screw tube (30 mm in diameter) was fastened and sealed to the wall at the same distance from the lower edge of the screw cap. To one of the screw tubes was affixed a two-way flow membrane air filter (0·22 µm) (Pall Australia), while the second was fitted with a removable sealed cap for medium inoculation.
A PVC tube (500 mm long, 50 mm in diameter) was centrally secured with 316 stainless steel locating pins inside the main reactor tube. The lower end of the central air-lift tube was located level with the bottom of the outside wall before fitting the conical section. The top of the central tube was located 10 cm lower than the surface level of the 10 L culture to prevent frothing. A sparging tube was fitted within the central tube 50 mm from the central tube base to deliver sterile air from a compressor through an in-line 0·22 µm membrane air filter (Pall Australia). An air pressure control valve (American Precision Valves, Richmond, CA, USA) was also fitted in-line before the filter to control the rate of air flow. The system was sterilized by infusion of an ethylene oxide and carbon dioxide gas mixture (90:10) by volume through the lower drainage tap for 24 h. The sterilizing gas was easily purged from the system by a vacuum pump, allowing sterile replacement air to enter via the two-way air filter.
Mini-bioreactor production of phage
Streptomyces scabies AS17 was used as the propagation host for ØAS1. The host was grown on OMY plates and incubated in the dark for 10 days. Spores were harvested by scraping the surface of 20 plates into 1 L of sterile 10% glycerol and storing at −20°C. Four mini-bioreactors were each filled with 10 L of sterile PYCa broth into which 200 mL of propagation host suspension (1 × 1010 cfu mL−1) and 200 mL of phage suspension (1 × 1012 PFU mL−1) were inoculated. The suspension was incubated in the dark for 4 days. After incubation, the suspension was centrifuged for 1 h at 480 g and serially filtered through decreasing pore-size filters culminating in a 0·22 µm tubular membrane filter (Pall Australia). After filtration, the phage suspension was calculated to be 1 × 109 PFU mL−1 by serial dilution. A total of 40 L of phage suspension was produced and stored at 4°C.
Phage bath design and construction
To circulate the phage suspension and maximize infection of S. scabies on infected tubers, a purpose-built phage bath was designed (Fig. 2). Two of these were constructed. The outer casing was a stainless steel trough (260 × 260 × 450 mm) with a lid. A smaller stainless steel trough (250 × 100 × 230 mm) was attached lengthways and centrally to the inside of the casing. The base of the smaller trough was raised 50 mm above the base of the outer casing by stainless steel legs welded into position. The base of the inner trough was fitted with a stainless steel mesh base (6 mm mesh size) attached a further 50 mm above the lower wall height of the inner trough. Attached directly below the mesh base, a stainless steel air-sparging pipe (20 mm in diameter) was fitted lengthways and centrally. The tube had a 90° bend at one end and was sealed at the other. Twenty-two 2 mm holes were drilled evenly and centrally at 10 mm spacings along the uppermost surface of the pipe section located below the mesh base. The upward bend of the pipe extended above the height of the outer casing by 50 mm. An adjustable air-flow control valve (American Precision Valves, USA) was fitted to the upper pipe end to control the rate of air flow and lift within the phage suspension. Air was supplied directly from an air compressor. When full, the top of the inner trough was below the surface level of the phage suspension to minimize potential frothing. The valve-controlled air-lift was enough to circulate the suspension gently.
Phage treatment of diseased tubers
One hundred and twenty first-progeny diseased tubers from the pathogenicity experiment were randomly divided into two equal groups and used as seed tubers. Group 1 was treated with phage by placing tubers into the inner trough of the bath of circulating prepared phage suspension at 28°C (± 2°C) for 24 h. Group 2 was treated in the same way with an identical suspension that had been autoclaved at 121°C for 20 min and used as the control. A controlled rate of air flow from a compressor through the air-sparging pipe gave a gentle rate of air-lift sufficient to circulate the suspension evenly among tubers. Treated tubers were planted at a depth of ≈ 5 cm into identical free-draining polystyrene boxes, as used in the production of diseased seed tubers containing steam-sterilized field soil, and watered and treated as described previously for the production of diseased seed tubers. Each treatment was replicated 10 times with six plants per replicate in fully randomized blocks. The experiment was repeated once.
Second-progeny tubers were harvested 8 weeks after commencement of the experiment. Leaves were removed and tubers inspected for disease. Scab lesions per potato tuber were counted and severity assessed as percentage lesion surface area and lesion type using a modification of the scale described by Loria & Kempter (1986). Lesion surface area (%) was rated on a scale of 0–6 as follows: 0, no lesions; 1, ≤ 10%; 2, 11–20%; 3, 21–30%; 4, 31–40%; 5, 41–50%; 6, > 50%. Lesion type was rated on a scale of 0–4 as follows: 0, no lesions; 1, superficial lesions; 2, slightly raised or slightly pitted lesions; 3, pitted lesions; 4, deeply pitted lesions. Weight, size and number of tubers per plant were examined to determine differences between phage-treated and nonphage-treated controls.
A randomized complete block design was used and analysis of variance was carried out using Superanova® (Abacus Concepts, Inc., Berkeley, CA, USA) to evaluate the effect of ØAS1 and S. scabies AS17 on the development of common scab disease symptoms. Significant differences between means were determined by Fisher's protected LSD test at P = 0·05.
Results and discussion
Isolation and identification of streptomycetes associated with common scab lesions on potato
Eighteen suspected streptomycete colonies were observed after 14 days of growth on starch-casein agar. Only one isolate, AS17, produced spiral spore chains and melanin pigments on both peptone-yeast-extract-iron agar and tyrosine agar and was tentatively identified as S. scabies (Table 1) using the criteria described by Lambert & Loria (1989). In addition, conversion of tyrosine to melanin by isolate AS17 on tyrosine agar stimulated rapid sporulation and made a purification step via OMY medium redundant.
Table 1. Characteristics of Streptomyces scabies (isolate AS17)
Phage ØAS1 selected to inhibit S. scabies formed small circular clear plaques on agar cultures, 1·5 mm in diameter, after 48 h. Negatively stained ØAS1 fitted the Siphoviridae (B1) morphotype (Francki et al., 1991) and consisted of an icosahedral head 40·0 nm long and 45·0 nm in diameter, with a tail 6 nm wide and 150 nm long.
Addition of phage ØAS1 to a 0·2 mL suspension of 1 × 1012 PFU mL−1 lysed S. scabies AS17 on a PYCa plate after 48 h of incubation in the dark showing a clear circular zone of ≈ 33 mm in diameter (Fig. 3). Four other accessions of S. scabies from the American Type Culture Collection and eight other Streptomyces species were susceptible to lysis by phage ØAS1. Twenty-four other species were not susceptible (Table 2).
Table 2. Host range of Streptomyces phage ØAS1 isolated from potato-cultivated soil
Streptomyces phage (ØAS1)
ACM, Australian Collection of Microorganisms; CBS, Centraalbureau voor Schimmelcultures, Baarn, The Netherlands; ATCC, American Type Culture Collection, Rockville, Maryland; NCTC, National Collection of Type Cultures, London, UK; +, host species susceptible to phage lysis; –, species not susceptible to phage lysis.
+ (propagation host)
Phages attacking Streptomyces spp. are widespread in the soil environment and readily detected (Williams & Lanning, 1984). The isolated phage (ØAS1) corresponded with the morphology of a Streptomyces scabies phage described by Ogiso et al. (1999) and its broad spectrum of activity was similar to descriptions by Wellington & Williams (1981). Common phage susceptibility is a distinctive characteristic of the genus Streptomyces and probably aids phage survival and replication in soil (Williams & Lanning, 1984). The phage ØAS1 appeared to be specific to certain Streptomyces species as it did not transgress the boundaries of the genus. This reflected the findings of Wellington & Williams (1981) where adsorption of Streptomyces phage was restricted to the cell wall chemotype 1 of the family Streptomycetaceae (Prauser, 1970). In the streptomycete host, AS17 phage replication occurred without concomitant host cell division.
Phage treatment of diseased tubers
A total of 212 infected first-progeny tubers were produced from the initial mini-tuber plantings, of which 120 were randomly selected for the phage treatment.
Untreated second-progeny tubers had a mean level of common scab lesion coverage of 23·0% compared with a significantly (P < 0·05) reduced lesion coverage (1·2%) in the progeny of phage-treated tubers (Table 3). In addition, lesion type and number of lesions per tuber were also significantly (P < 0·05) reduced by phage treatment (Table 3). Isolate AS17 was recovered from infected potatoes of both treatments using starch-casein agar amended with nystatin and cycloheximide. No significant differences were recorded in tuber weight (Table 3), size or number between phage-treated and untreated tubers. Data from repeated experiments were not significantly different and accordingly were pooled for analysis.
Table 3. Effect of Streptomyces phage (ØAS1) application on number of lesions, lesion surface area and lesion type of Streptomyces scabies (isolate AS17) common scab disease in second-progeny potato tubers under glasshouse conditions
Lesion type was rated on a scale of 0–4 as follows: 0, no lesions; 1, superficial; 2, slightly raised or slightly pitted; 3, pitted; 4, deeply pitted.
Values followed by the same letter within a column are not significantly (P > 0·05) different according to Fisher's protected LSD test. Results are means of 10 replicates for each treatment.
Non-phage-treated second progeny
Phage-treated second progeny
This study is the first in which a Streptomyces phage has been used as a biocontrol agent in vivo to disinfest potato seed tubers of common scab disease, thus minimizing the risk of contaminating noninfested soil with tuber-borne inoculum. In a trial of this size, the large volume of phage suspension required, and the filamentous nature and the associated clumping of the host (Dowding, 1973) were overcome by the design of the simple mini-bioreactor. Clumping of mycelium was not evident and the phage suspension showed high lytic activity. In this study, phage and actinomycete host cells were added simultaneously to the bioreactor as host cells may be more susceptible to lysis at an early stage of their growth/reproductive cycle (Patten et al., 1995). However, the optimum air-lift conditions and host/phage inoculum ratio remain to be established.
In the present study scab-affected tubers were treated in the phage bath for 24 h. The bath allowed the same constant movement of phage suspension as the mini-bioreactor. A further modification permitting movement of tubers concurrently with phage suspension is currently under design. This may eliminate host persistence where scab lesions may have been protected from phage adsorption by tuber–tuber contact. The inoculum of the phage in this study was lodged on the scab-infested mother tubers and significantly reduced the incidence and severity of common scab on the progeny tubers grown in steam-pasteurized soil. The novel method of phage propagation by an inexpensive and effective mini-bioreactor and subsequent treatment in the air-lift phage bath appears to be unique and provides the potential to disinfest large quantities of seed potato tubers at low cost before planting.
Future studies for screening known strains of common scab-causing Streptomyces spp. for sensitivity to this and other phages would indicate the potential use of this mechanism on a larger scale. In addition, field trials will determine the efficacy of this phage treatment when phage-disinfested tubers are grown in soil containing a large reservoir of soil-borne inoculum.
The authors thank Professor K. Sivasithamparam for his valuable discussions and for reading the manuscript. We also thank the United Arab Emirates University for their cooperation and participation in this study.