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In vitro screening for the identification of potential biocontrol agents of Allium white rot
430
Mycol. Res. 95 (4): 430-434 (1991) Printed in Great Britain
In vitro screening for the identification of potential biocontrol agents of Allium white rot
A. M. JACKSON, J. M. WHIPPS AND J. M. LYNCH AFRC Institute of Horticultural Research, Uttlehampton, West Sussex, BN17 6LP, U.K
A multi-test in vitro system was designed to identify antagonistic micro-organisms of the Allium white rot (AWR) pathogen, Sclerotium cepivorum. Micro-organisms (215) comprising 140 fungi, 58 bacteria and 17 actinomycetes were isolated from AWR soils, pathogen sclerotia and diseased host tissue. Fungi were screened and scored for volatile and non-volatile antibiotic activity, inhibition of sclerotium formation, and overgrowth of colonies of S. cepivorum on agar plates. Bacteria and actinomycetes were screened for antibiotic activity only. Comparisons with known antagonists of plant pathogens were also made. The screens enabled selection of antagonists to be made based on either a 'total score' from all tests or on a potential mode of action basis in selected tests. Statistical analysis of total score identified inhibition of sclerotia formation and position of sclerotia formation in dual culture as the most discriminating variates in the screen.
Allium white rot (AWR) caused by the soil-borne fungus Sclerotium cepivorum Berk. is one of the most destructive and difficult diseases of onion to control. Sclerotia of S. cepivorum germinate myceliogenically in the presence of Allium roots and mycelial infection of the roots subsequently occurs (Coley-Smith, 1960). Later in the season the disease spreads between plants in rows, causing epidemics (Entwistle & Munasinghe, 1978). Seed and stem base applications of vinclozolin and iprodione are most commonly used for AWR control on salad onions but are less effective on dry bulb onions (Entwistle, 1983). Loss in effectiveness of these fungicides has been reported (Entwistle, 1986), apparently due to enhanced microbial degradation of these chemicals in the soil rather than fungicide resistance in the pathogen (Walker, Brown & Entwistle, 1986). There are no alternative methods for large-scale AWR control. The disease results in uneconomic yields four years after its introduction in successive onion cropping (Coley-Smith, 1987). The use of antagonistic micro-organisms is a possible alternative to chemical control of AWR. However, past attempts to identify antagonists for use as biocontrol agents have frequently failed (Entwistle, 1988). This could be due to inappropriate design of the in vitro screening systems, often based on a single test. With this in mind, we developed a multi-test primary screening system to identify potential antagonists, so that organisms could be ranked for total antagonistic activity and also for individual modes of action. We then used multivariate analysis to identify the contribution of different assay results to the total score for antagonism.
MATERIALS AND METHODS Test isolates Potential antagonists were isolated from healthy and white rot infected Allium bulbs and white rot infested soils kindly provided from throughout the U.K. by the Agricultural Development and Advisory Service (ADAS) and other research workers. In addition, antagonistic micro-organisms that had previously shown some activity against Sclerotium cepivorum and other sclerotium-forming pathogens were used as internal standards. A total of 215 isolates were screened in vitro to identify those with antagonistic activity against the mycelium of the white rot pathogen. For isolation of antagonists, onions infected with AWR were stored at 5 °C for 24 h to allow the development of fungal mycelium. Fifty sclerotia were removed, where possible, and placed onto moist filter paper in a Petri dish and incubated for up to two months at 15°. Sclerotia were observed routinely and any spores or mycelia that developed on the sclerotium surface were transferred to Oxoid potato dextrose agar (PDA). Mycelium present on the bulbs was transferred onto PDA supplemented with Aureomycin (0'32 g 1-1 of a wettable powder containing 5'5 % chlortetracycline HCl; Cyanamid) to give 17'6 mg a.i. 1-1. For isolation from stem tissue or soil adhering to the stem (bulb) base, 2 g of the appropriate material was macerated in 25 ml sterile distilled water in a Waring blender for 15 sec. Dilution plates were prepared on PDA. Fungal isolates were sub-cultured onto fresh PDA by hyphal tip transfer and bacteria by streaking out onto Oxoid nutrient agar. Actinomycetes were isolated from four white rot infested soils using the soil dilution plate methods of Athalye & Goodfellow (1981) and Dhingra & Sinclair (1986).
A M. Jackson, J. M. Whipps & J. M. Lynch Fungi, bacteria and actinomycetes were stored on slopes of PDA, nutrient agar and R2YE medium (Hopwood et al., 1985), respectively. The most active cultures in the in vitro screen were also transferred to liquid nitrogen (Challen & Elliott 1986), as was the standard isolate of Sclerotium cepivorum (9181) from A Entwistle, IHR-Wellesbourne, U.K. Identities of fungi showing high activity against the white rot pathogen in vitro were confirmed at C.AB. International Mycological Institute. Bacteria were characterised by physiological and biochemical properties, using API 20 B galleries (API-bioMerieux Ltd., UK), and by cell morphology, Gram reaction, spore production, oxygen requirements and colony characteristics. The actinomycetes were not identified.
Screening systems Fungi. An in vitro screening system on PDA was based on the tests used by Dennis & Webster (1971a, b, c) and Whipps (1987). It was designed to identify antagonists that produce non-volatile or volatile antibiotics or that are potential mycoparasites. In addition, inhibition of sclerotia formation was assessed. For a few isolates, the tests were also done on tap water agar containing 15 g Oxoid Technical Agar no. 3 1- 1 . Each test was replicated three times for each isolate, at 15°, and every batch of tests included two known antagonists, Coniothyrium minitans (IMI 134523) and Gliocladium virens (G20), as internal standards. Non-volatile antibiotic production was estimated by placing a 5 mm inoculum disc of antagonist centrally on cellophanecovered agar and after 3 days the antagonist and cellophane were removed. A 5 mm disc of the pathogen was then placed centrally on the agar and pathogen growth rate was recorded over 3 days. To determine whether complete inhibition of growth was due to the production of a fungistatic or
Fig. 1. Generalized diagram to show positions of sclerotia formation in dual-culture tests with fungi. Four zones (l-4) within the Sclerotium cepivorum colony were defined. X indicates position of inoculation of antagonist and S. cepivorum.
431 fungitoxic antibiotic the pathogen inoculum disc was transferred to fresh PDA medium and examined for renewed growth. The production of volatile antibiotics was studied using Petri dish bases sealed to one another. The uppermost plate received a 5 mm disc of S. cepivorum and the bottom plate was inoculated Simultaneously with the test isolate. The percentage inhibition of growth was calculated by comparing growth of S. cepivorum in the presence of a test isolate with growth in controls. Dual culture plates were used initially to assess inhibition of radial growth of the pathogen using the technique of Royse & Ries (1978); the antagonist and pathogens were inoculated simultaneously 4 cm apart. The plates were also used to record the type of colony interaction. The time to sclerotia formation was noted for both immature 'white' sclerotia and mature 'black' sclerotia. The position of sclerotia formation (Fig. 1) was recorded after 6 days for white sclerotia and 13 days for black sclerotia. Percentage inhibition of sclerotia formation was determined after 13 days by counting numbers of sclerotia in a 1 cm 2 area (zone 1, Fig. 1) on the edge of the pathogen colony between the inoculation points, compared with a similar position on control plates inoculated with two discs of the pathogen.
Bacteria and actinomyeetes. Bacteria and actinomycetes were screened as for fungi but NA was used instead of PDA which did not support the growth of some isolates of bacteria. However, on nutrient agar pathogen sclerotia did not develop and so the overall activity of each bacterial isolate or actinomycete was based on three determinations only: nonvolatile and volatile antibiotic tests and dual-culture inhibition. Co-inoculation plates were produced by applying a cell suspension as an off-centred single streak, 4 em from the pathogen inoculum disc. In volatile and non-volatile antibiotic tests the inoculum was placed in the centre of an agar plate, with an inoculating loop to produce a colony of 2 cm diameter. As some bacteria produced cellulase, the cellophane disc and bacterial isolate in non-volatile antibiotic tests were removed after 2 days instead of 3 days as for actinomycetes and fungi. Also, actinomycetes were grown on test plates for 2 days at 30° prior to inoculation with the pathogen as there was poor growth at 15°. Scoring and classification of activities Each isolate was given a score for overall activity in the tests (Table 1). For fungi it was based on seven assessments in dual culture tests and Single assessments in the volatile and nonvolatile antibiotic tests. Each assessment was rated from 0 to 10, giving a maximum obtainable score of 90. A percentage total score (T score) was obtained. Isolates with the higher scores were the more antagonistic. The scoring of bacteria and adinomycetes was based on one assessment in each of the three tests. The maximum and minimum scores were 30 and o respectively, and percentage total scores were calculated. Using the scores from individual tests, isolates were classed on a mode of action basis as good, intermediate or poor antibiotic producers. In addition, fungi were also classed as good, intermediate or poor' mycoparasites ' on the assumption
Screening for Allium white rot biocontrol agents
432
Table 1. Description of the scoring system used in primary screening tests against Sclerotium cepivorum Factors scored
% inhibition
type b
of sclerotia formation (13 days)
Time (d) to sclerotia formation (dual culture)
Position of sclerotia formation'
White
Black
PosWS & PosBS
7
> 10
6 5
10 9 8 7 6
Interaction
Percent inhibition'
Code
NVA
VA& DC
Inter
Form
Score 10 9 7 5 3 1 o
loot 100' 50-99 30-49 10-29 1-9
90-100 70-89 50-69 30-49 10-29 1-9
1.4
100 90-99 70-89 50-69 30-49 10-29
<=0
<=0
1/2,3,2/1 2
4 3 2
4
43 42 432 431 4321
0-9
• Percentage inhibition of S. cepivorum mycelium in volatile antibiotic test (VA), non-volatile antibiotic test (NVA) or dual-culture test (DC); t = no hyphal re-growth after transfer of inoculum to fresh PDA; " hyphal re-growth after transfer of inoculum to fresh PDA. b 1, antagonist overgrowing pathogen and pathogen stopped; 1/2, antagonist overgrowing pathogen but pathogen still growing; 2, pathogen overgrowing antagonist and antagonist stopped; 2/1, pathogen overgrowing antagonist but antagonist still growing; 3, mutual inhibition 1-3 mm; 4, extreme inhibition> 4 mm. , After 6 days for white sclerotia (PosWS) and 13 days for black sclerotia (PosBS) in zones 1-4 (see Fig. 1).
that overgrowth of the pathogen can be indicative of mycoparasitic behaviour in terms of potential nutrient acquisition (sensu Lewis, Whipps & Cooke, 1989).
RESULTS Micro-organisms isolated The 215 micro-organisms obtained consisted of 140 fungi, 58 bacteria and 17 actinomycetes. The most frequently occurring isolates of fungi were Trichoderma spp. (14) and Penicillium spp. (8). Species of Pseudomonas or Alcaligenes were isolated at the same frequency as for Trichoderma. Eight isolates of Enterobacteriaceae were the next most common group of bacteria isolated and had a similar frequency of occurrence to Penicillium spp.
In vitro screening of test organisms Fungi. In volatile antibiotic tests no fungi inhibited mycelial growth of the pathogen by 25 % or more. In non-volatile tests 12 (8'6%) fungal isolates gave complete inhibition by
producing fungitoxic metabolites and 36 (25'7%) produced fungistatic metabolites. Complete inhibition of sclerotia formation on the periphery of the pathogen colony closest to the test isolate was achieved by 79 (56'4 %) fungal isolates. The main scoring system was used to rank isolates in order of their overall activity in primary screening tests. The T scores of fungi ranged from 20 to 78 (Table 2). Twenty-six fungal isolates obtained scores of 70 or above. All isolates had poor volatile antibiotic activity. In general, formation of sclerotial initials occurred after 6 days and sclerotia maturation was complete after 9 days on control and dual culture plates. In dual culture, the internal standard G. virens (G20) and T. viride (IMI 322663) had most effect on formation and maturation of sclerotia: sclerotial initials occurred on the colony periphery (zones 4, 3 and I; see Fig. I), but those on the colony edge closest to the antagonist (zone I) were prevented from maturing, whilst those on the colony edge furthest from the antagonist (zones 4 and 3) matured. All Trichoderma spp among fungi with the top six T scores were capable of overgrowing the pathogen colony. Isolates were also placed into groups based on modes of action (Table 3). Twenty-seven (19'3 %) fungal isolates were good mycoparasites. This group occurred more frequently
Table 2. Number of isolates in each T score range T score range
Fungi
90-100 80-89 70-79 60-69 50-59 40-49 30-39 20-29 10-19 0-9
0 0 26 32 43 28 9 2 0 0
Bacteria
Actinomycetes
0 0 3
0 4 1
5 15 10
5 1 1 1 2 1 1
12
7 4 2
Table 3. Number of isolates obtained in each mode of action group Activity group
Microbial group
Mode of action
Good
Intermediate
Poor
Fungi
Antibiosis 'Mycoparasitism' Both Antibiosis Antibiosis
13 27 27 3 8
113 6 18 30 4
14 107 95 25 5
Bacteria Actinomycetes
A. M. Jackson, J. M. Whipps & J. M. Lynch
than good antibiotic producers but often strains exhibiting 'mycoparasitism' also had some antibiotic-producing capability. Most fungal isolates were poor mycoparasites or intermediate antibiotic producers.
Bacteria and actinomycetes. In volatile antipiotic tests,
433
T score but the top 60 isolates in both PCA ranking and T score ranking were defined by near maximum scores for Form and PosBS. Subsequent PCA on these top 60 isolates gave a different ranking order to that obtained by PCA of all isolates and further indicated that within the top 30 isolates there was little discrimination in the T score system.
4 (6'9%) bacteria and 6 (35'3%) actinomycetes inhibited
mycelial growth of the pathogen by 25 % or more. Six (10'3 %) bacteria and 3 (17'6%) actinomycetes were fungitoxic to the pathogen and 14 (24'1 %) bacteria and 3 (17'6%) actinomycetes were fungistatic. The T scores of the bacteria ranged from 0 to 77 (Table 2). The four most active isolates (T scores of 67 to 77) were Pseudomonas or Alcaligenes spp. Of the 8 most active isolates, one had volatile antibiotic activity which inhibited mycelial growth by 41 %,6 produced fungitoxic non-volatile antibiotics and 2 produced fungistatic non-volatile antibiotics. T scores of actinomycetes ranged from 3 to 87. The highest-ranked isolate inhibited pathogen growth by 89% by the production of volatile antibiotics. Of the top 10 isolates with scores between 60 and 90, two produced fungistatic and three produced fungitoxic non-volatile antibiotics. In dual culture, pathogen growth was inhibited by 55 to 100%.
Multivariate analysis of the T score system To determine the relative contribution of assessments to the T score for fungi, a multivariate analysis using principal component analysis (PCA) was done (Morrison, 1967). Because the tests had different ranges, a correlation rather than a covariance matrix of the data was used. Negative values in the NVA. VA. DC and Form tests (see Table 1) were censored to zero. The multivariate analysis showed that T score was most highly correlated with PosBS and Form (Table 4) and the first principal component, which was essentially a combination of these two variates, accounted for 29% of the total variation. The first principal component score had a correlation of 0'882 with T score, 0'860 with PosBs and 0'825 with Form. The second principal component, which accounted for 24 % of the variation, was a simple mean of NVA. DC and Inter. Correlation of this second component with T score was poor as this component represented variation not accounted for by the first component which was closely related to T score. Overall the general ranking derived from PCA was similar to
Table 4. Major correlations (r) between T score and individual screening tests obtained using multivariate analysis Interactions' T score PosBS T score Inter T score PosBS NVA T score T score • See Table 1 for codes.
28
r value v. PosBS v. Form v. Form v.NVA v. Inter v. PosWS v. DC v. PosWS v. NVA
0'784 0'730 0'716 0'675 0'633 0'598 0'556 0'519 0'517
DISCUSSION The primary screening system included observations on the effects of micro-organisms on mycelial growth and sclerotia formation of Sclerotium cepivorum. It quantified isolate activity and provided some information on modes of action. Actinomycetes and bacteria produced both volatile and non-volatile antibiotics whereas the fungi only produced nonvolatile antibiotics that severely inhibited pathogen growth. Various effects on sclerotial formation were observed in response to test micro-organisms. Some isolates prevented the formation of sclerotial initials whereas others prevented their maturation, perhaps by blocking the metabolic pathway to pigment production. The availability of nutrients can influence sclerotial initial formation and sclerotia pigmentation (Townsend, 1957) and fast-growing isolates such as Trichoderma spp. may act in this way. Some fungi had no effect on sclerotial formation and maturation but in 40 % of cases the number of sclerotia was significantly lower than on control plates, indicating some more subtle interaction. Fungi, bacteria and actinomycetes have been tested before in dual-culture against S. cepivorum but an objective of this study was to design a screening system to enable modes of action to be distinguished as well as producing a general ranking of antagonism. For example, some Trichoderma isolates were highly effective in antibiosis whereas others were comparatively poor antibiotic producers. However, some of the latter invaded host colonies very rapidly, indicative of mycoparasitism which some previous screens may have neglected. However, statistical analyses of the T score system indicated that percent inhibition of sclerotia formation (Form) and position of black sclerotia formation (PosBS) formed the basic structure of the ranking, after which non-volatile antibiotic (NVA) and interaction type (Inter) assessments could be used for further discrimination. Nevertheless, these four assessments were not sensitive enough to differentiate the ranking in the top 30 isolates. So a cheaper and more efficient primary screen would have been achieved by considering just the initial two variates, Form and PosBS, and carrying out further screening on isolates with high scores in these alone. Interaction tests would be used in the next level of screening and would detect mycoparasitic potential. Indeed without interaction tests or VA tests (which were not discriminating in the T score system), 'mycoparasitism' and the potent volatile antibiotic produced by one of the actinomycetes would not have been detected. A multi-test screening system thus has advantages over a one- or two-test system if it represents several different modes of action. This approach to the selection of potentially useful antagonists for control of AWR may be of interest to plant pathologists in general. Importantly, when isolates with scores from throughout the T score range were tested subsequently in an in vivo MYC 95
434
Screening for Allium white rot biocontrol agents bioassay of Entwistle & Spence (1988) there was a close association between the results of these in vitro and in vivo tests (A. R. Entwistle & J. Clarkson, in preparation).
We thank Drs A. R. Entwistle and R. T. Burchill (IHRWellesbourne) for their co-operation, John Fenlon and Andrew Mead for help with statistical analyses and experimental design and the Agricultural Genetics Company for financial support. We would also like to thank the many research workers throughout the world who provided fungal and bacterial isolates for use in this screen.
REFERENCES Athalye, M. &< Goodfellow. M. (l981). Selective isolation and enumeration of actinomycetes using Rifampicin. Journal ofApplied Bacteriology 51. 289-297. Challen, M. P &< Elliott. T. J. (1986). Polypropylene straw ampoules for the storage of micro-organisms in liquid nitrogen. Journal of Microbiological
Methods 5, 11-23. Coley-Smith. J. R. (1960). Studies of the biology of Sclerotium cepivorum Berk. IV. Germination of sclerotia. Annals of Applied Biology 48. 8-18. Coley-Smith, J. R. (1987). Alternative methods of controlling white rot disease of Allium. In Innovative Approaches to Plant Disease Control (ed. Chet. I.). pp. 161-177. New York. USA: John Wiley. Dennis, C. &< Webster. J. (1971 a). Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Transactions of the
British Mycological Society 57, 25-39. Dennis. C. &< Webster, j. (1971 b). Antagonistic properties of species-groups of Trichoderma. II. Production of volatile antibiotics. Transactions of the
British Mycological Society 57, 41-48. Dennis. C. &< Webster, J. (l971 c). Antagonistic properties of species-groups of Trichoderma. III. Hyphal interaction. Transactions of the British Mycological
Society 57. 363-369. Received for publication 16 February 1990 and in revised form 24 July 1990
Dhingra. O. D. 8< Sinclair. J. B. (1986). Basic Plant Pathology Methods, pp. 285-318. Boca Raton, Florida, USA: CRC Press. Entwistle, A R. (1983). Needs and outlook for chemical control. In Proceedings of the Second International Workshop on Allium White Rot, pp. 20-22. Beltsville, MD. Entwistle. A R. (1986). Loss of control of Allium white rot by fungicides and its implications. Aspects of Applied Biology 12. 201-209. Entwistle, A R. (1988). Opportunities for the microbial control of Allium white rot. EPPO Bulletin 18, 19-28. Entwistle, A R. 8< Munasinghe. H. L. (1978). Epidemiology and control of white rot disease of onions. In Plant Disease Epidemiology (ed. Scott. P. R. &< Bainbridge. A). pp. 187-191. Oxford, U.K.: Blackwell Scientific Publications. Entwistle, A R. &< Spence. N. J. (1988). Progress in the development of a standard method for measuring resistance in Allium to white rot (Sclerotium cepivorum). In 4th Eucarpia Allium Symposium (ed. Riggs. T. J.• Astley, D.• Brewster. J. L.. Dawson. P. R. &< Maude, R. B.). pp. 29-37. Wellesbourne. U.K.: Institute of Horticultural Research. Hopwood, D. A. Bibb, M. J.• Chater, K. F., Kieser. T., Bruton, C. L Kieser. H. M.. Lydiate. D. L Smith, C. P. 8< Ward, j. M. (1985). Genetic Manipulation of Streptomyces: A Laboratory Manual. Norwich, UK: The John Innes Foundation. Lewis, K., Whipps. J. M. 8< Cooke, R. C. (l989). Mechanisms of biological disease control with special reference to the case study of Pythium oligandrum as an antagonist. In Biotechnology of Fungi for Improving Plant Growth (ed. Whipps, J. M. &< Lumsden, R. D.), pp. 191-217. Cambridge University Press. Morrison, D. F. (1967). Multivariate Statistical Methods. New York: McGrawHill. Royse, D. L. &< Ries, S. M. (1978). The influence of fungi isolated from peach twigs on the pathogenicity of Cytospora cincta. Phytopathology 68, 603-607. Townsend. B. (1957). Nutritional factors influencing the production of sclerotia of certain fungi. Annals of Botany 21. 153-166. Walker. A. Brown. P. A &< Entwistle, A R. (1986). Enhanced degradation of iprodione and vinclozolin in soil. Pesticide Science 117. 183-193. Whipps, J. M. (1987). Effect of media on growth and interactions between a range of soil-borne glasshouse pathogens and antagonistic fungi. New
Phytologist 107. 127-142.
Mycol. Res. 95 (4): 430-434 (1991) Printed in Great Britain
In vitro screening for the identification of potential biocontrol agents of Allium white rot
A. M. JACKSON, J. M. WHIPPS AND J. M. LYNCH AFRC Institute of Horticultural Research, Uttlehampton, West Sussex, BN17 6LP, U.K
A multi-test in vitro system was designed to identify antagonistic micro-organisms of the Allium white rot (AWR) pathogen, Sclerotium cepivorum. Micro-organisms (215) comprising 140 fungi, 58 bacteria and 17 actinomycetes were isolated from AWR soils, pathogen sclerotia and diseased host tissue. Fungi were screened and scored for volatile and non-volatile antibiotic activity, inhibition of sclerotium formation, and overgrowth of colonies of S. cepivorum on agar plates. Bacteria and actinomycetes were screened for antibiotic activity only. Comparisons with known antagonists of plant pathogens were also made. The screens enabled selection of antagonists to be made based on either a 'total score' from all tests or on a potential mode of action basis in selected tests. Statistical analysis of total score identified inhibition of sclerotia formation and position of sclerotia formation in dual culture as the most discriminating variates in the screen.
Allium white rot (AWR) caused by the soil-borne fungus Sclerotium cepivorum Berk. is one of the most destructive and difficult diseases of onion to control. Sclerotia of S. cepivorum germinate myceliogenically in the presence of Allium roots and mycelial infection of the roots subsequently occurs (Coley-Smith, 1960). Later in the season the disease spreads between plants in rows, causing epidemics (Entwistle & Munasinghe, 1978). Seed and stem base applications of vinclozolin and iprodione are most commonly used for AWR control on salad onions but are less effective on dry bulb onions (Entwistle, 1983). Loss in effectiveness of these fungicides has been reported (Entwistle, 1986), apparently due to enhanced microbial degradation of these chemicals in the soil rather than fungicide resistance in the pathogen (Walker, Brown & Entwistle, 1986). There are no alternative methods for large-scale AWR control. The disease results in uneconomic yields four years after its introduction in successive onion cropping (Coley-Smith, 1987). The use of antagonistic micro-organisms is a possible alternative to chemical control of AWR. However, past attempts to identify antagonists for use as biocontrol agents have frequently failed (Entwistle, 1988). This could be due to inappropriate design of the in vitro screening systems, often based on a single test. With this in mind, we developed a multi-test primary screening system to identify potential antagonists, so that organisms could be ranked for total antagonistic activity and also for individual modes of action. We then used multivariate analysis to identify the contribution of different assay results to the total score for antagonism.
MATERIALS AND METHODS Test isolates Potential antagonists were isolated from healthy and white rot infected Allium bulbs and white rot infested soils kindly provided from throughout the U.K. by the Agricultural Development and Advisory Service (ADAS) and other research workers. In addition, antagonistic micro-organisms that had previously shown some activity against Sclerotium cepivorum and other sclerotium-forming pathogens were used as internal standards. A total of 215 isolates were screened in vitro to identify those with antagonistic activity against the mycelium of the white rot pathogen. For isolation of antagonists, onions infected with AWR were stored at 5 °C for 24 h to allow the development of fungal mycelium. Fifty sclerotia were removed, where possible, and placed onto moist filter paper in a Petri dish and incubated for up to two months at 15°. Sclerotia were observed routinely and any spores or mycelia that developed on the sclerotium surface were transferred to Oxoid potato dextrose agar (PDA). Mycelium present on the bulbs was transferred onto PDA supplemented with Aureomycin (0'32 g 1-1 of a wettable powder containing 5'5 % chlortetracycline HCl; Cyanamid) to give 17'6 mg a.i. 1-1. For isolation from stem tissue or soil adhering to the stem (bulb) base, 2 g of the appropriate material was macerated in 25 ml sterile distilled water in a Waring blender for 15 sec. Dilution plates were prepared on PDA. Fungal isolates were sub-cultured onto fresh PDA by hyphal tip transfer and bacteria by streaking out onto Oxoid nutrient agar. Actinomycetes were isolated from four white rot infested soils using the soil dilution plate methods of Athalye & Goodfellow (1981) and Dhingra & Sinclair (1986).
A M. Jackson, J. M. Whipps & J. M. Lynch Fungi, bacteria and actinomycetes were stored on slopes of PDA, nutrient agar and R2YE medium (Hopwood et al., 1985), respectively. The most active cultures in the in vitro screen were also transferred to liquid nitrogen (Challen & Elliott 1986), as was the standard isolate of Sclerotium cepivorum (9181) from A Entwistle, IHR-Wellesbourne, U.K. Identities of fungi showing high activity against the white rot pathogen in vitro were confirmed at C.AB. International Mycological Institute. Bacteria were characterised by physiological and biochemical properties, using API 20 B galleries (API-bioMerieux Ltd., UK), and by cell morphology, Gram reaction, spore production, oxygen requirements and colony characteristics. The actinomycetes were not identified.
Screening systems Fungi. An in vitro screening system on PDA was based on the tests used by Dennis & Webster (1971a, b, c) and Whipps (1987). It was designed to identify antagonists that produce non-volatile or volatile antibiotics or that are potential mycoparasites. In addition, inhibition of sclerotia formation was assessed. For a few isolates, the tests were also done on tap water agar containing 15 g Oxoid Technical Agar no. 3 1- 1 . Each test was replicated three times for each isolate, at 15°, and every batch of tests included two known antagonists, Coniothyrium minitans (IMI 134523) and Gliocladium virens (G20), as internal standards. Non-volatile antibiotic production was estimated by placing a 5 mm inoculum disc of antagonist centrally on cellophanecovered agar and after 3 days the antagonist and cellophane were removed. A 5 mm disc of the pathogen was then placed centrally on the agar and pathogen growth rate was recorded over 3 days. To determine whether complete inhibition of growth was due to the production of a fungistatic or
Fig. 1. Generalized diagram to show positions of sclerotia formation in dual-culture tests with fungi. Four zones (l-4) within the Sclerotium cepivorum colony were defined. X indicates position of inoculation of antagonist and S. cepivorum.
431 fungitoxic antibiotic the pathogen inoculum disc was transferred to fresh PDA medium and examined for renewed growth. The production of volatile antibiotics was studied using Petri dish bases sealed to one another. The uppermost plate received a 5 mm disc of S. cepivorum and the bottom plate was inoculated Simultaneously with the test isolate. The percentage inhibition of growth was calculated by comparing growth of S. cepivorum in the presence of a test isolate with growth in controls. Dual culture plates were used initially to assess inhibition of radial growth of the pathogen using the technique of Royse & Ries (1978); the antagonist and pathogens were inoculated simultaneously 4 cm apart. The plates were also used to record the type of colony interaction. The time to sclerotia formation was noted for both immature 'white' sclerotia and mature 'black' sclerotia. The position of sclerotia formation (Fig. 1) was recorded after 6 days for white sclerotia and 13 days for black sclerotia. Percentage inhibition of sclerotia formation was determined after 13 days by counting numbers of sclerotia in a 1 cm 2 area (zone 1, Fig. 1) on the edge of the pathogen colony between the inoculation points, compared with a similar position on control plates inoculated with two discs of the pathogen.
Bacteria and actinomyeetes. Bacteria and actinomycetes were screened as for fungi but NA was used instead of PDA which did not support the growth of some isolates of bacteria. However, on nutrient agar pathogen sclerotia did not develop and so the overall activity of each bacterial isolate or actinomycete was based on three determinations only: nonvolatile and volatile antibiotic tests and dual-culture inhibition. Co-inoculation plates were produced by applying a cell suspension as an off-centred single streak, 4 em from the pathogen inoculum disc. In volatile and non-volatile antibiotic tests the inoculum was placed in the centre of an agar plate, with an inoculating loop to produce a colony of 2 cm diameter. As some bacteria produced cellulase, the cellophane disc and bacterial isolate in non-volatile antibiotic tests were removed after 2 days instead of 3 days as for actinomycetes and fungi. Also, actinomycetes were grown on test plates for 2 days at 30° prior to inoculation with the pathogen as there was poor growth at 15°. Scoring and classification of activities Each isolate was given a score for overall activity in the tests (Table 1). For fungi it was based on seven assessments in dual culture tests and Single assessments in the volatile and nonvolatile antibiotic tests. Each assessment was rated from 0 to 10, giving a maximum obtainable score of 90. A percentage total score (T score) was obtained. Isolates with the higher scores were the more antagonistic. The scoring of bacteria and adinomycetes was based on one assessment in each of the three tests. The maximum and minimum scores were 30 and o respectively, and percentage total scores were calculated. Using the scores from individual tests, isolates were classed on a mode of action basis as good, intermediate or poor antibiotic producers. In addition, fungi were also classed as good, intermediate or poor' mycoparasites ' on the assumption
Screening for Allium white rot biocontrol agents
432
Table 1. Description of the scoring system used in primary screening tests against Sclerotium cepivorum Factors scored
% inhibition
type b
of sclerotia formation (13 days)
Time (d) to sclerotia formation (dual culture)
Position of sclerotia formation'
White
Black
PosWS & PosBS
7
> 10
6 5
10 9 8 7 6
Interaction
Percent inhibition'
Code
NVA
VA& DC
Inter
Form
Score 10 9 7 5 3 1 o
loot 100' 50-99 30-49 10-29 1-9
90-100 70-89 50-69 30-49 10-29 1-9
1.4
100 90-99 70-89 50-69 30-49 10-29
<=0
<=0
1/2,3,2/1 2
4 3 2
4
43 42 432 431 4321
0-9
• Percentage inhibition of S. cepivorum mycelium in volatile antibiotic test (VA), non-volatile antibiotic test (NVA) or dual-culture test (DC); t = no hyphal re-growth after transfer of inoculum to fresh PDA; " hyphal re-growth after transfer of inoculum to fresh PDA. b 1, antagonist overgrowing pathogen and pathogen stopped; 1/2, antagonist overgrowing pathogen but pathogen still growing; 2, pathogen overgrowing antagonist and antagonist stopped; 2/1, pathogen overgrowing antagonist but antagonist still growing; 3, mutual inhibition 1-3 mm; 4, extreme inhibition> 4 mm. , After 6 days for white sclerotia (PosWS) and 13 days for black sclerotia (PosBS) in zones 1-4 (see Fig. 1).
that overgrowth of the pathogen can be indicative of mycoparasitic behaviour in terms of potential nutrient acquisition (sensu Lewis, Whipps & Cooke, 1989).
RESULTS Micro-organisms isolated The 215 micro-organisms obtained consisted of 140 fungi, 58 bacteria and 17 actinomycetes. The most frequently occurring isolates of fungi were Trichoderma spp. (14) and Penicillium spp. (8). Species of Pseudomonas or Alcaligenes were isolated at the same frequency as for Trichoderma. Eight isolates of Enterobacteriaceae were the next most common group of bacteria isolated and had a similar frequency of occurrence to Penicillium spp.
In vitro screening of test organisms Fungi. In volatile antibiotic tests no fungi inhibited mycelial growth of the pathogen by 25 % or more. In non-volatile tests 12 (8'6%) fungal isolates gave complete inhibition by
producing fungitoxic metabolites and 36 (25'7%) produced fungistatic metabolites. Complete inhibition of sclerotia formation on the periphery of the pathogen colony closest to the test isolate was achieved by 79 (56'4 %) fungal isolates. The main scoring system was used to rank isolates in order of their overall activity in primary screening tests. The T scores of fungi ranged from 20 to 78 (Table 2). Twenty-six fungal isolates obtained scores of 70 or above. All isolates had poor volatile antibiotic activity. In general, formation of sclerotial initials occurred after 6 days and sclerotia maturation was complete after 9 days on control and dual culture plates. In dual culture, the internal standard G. virens (G20) and T. viride (IMI 322663) had most effect on formation and maturation of sclerotia: sclerotial initials occurred on the colony periphery (zones 4, 3 and I; see Fig. I), but those on the colony edge closest to the antagonist (zone I) were prevented from maturing, whilst those on the colony edge furthest from the antagonist (zones 4 and 3) matured. All Trichoderma spp among fungi with the top six T scores were capable of overgrowing the pathogen colony. Isolates were also placed into groups based on modes of action (Table 3). Twenty-seven (19'3 %) fungal isolates were good mycoparasites. This group occurred more frequently
Table 2. Number of isolates in each T score range T score range
Fungi
90-100 80-89 70-79 60-69 50-59 40-49 30-39 20-29 10-19 0-9
0 0 26 32 43 28 9 2 0 0
Bacteria
Actinomycetes
0 0 3
0 4 1
5 15 10
5 1 1 1 2 1 1
12
7 4 2
Table 3. Number of isolates obtained in each mode of action group Activity group
Microbial group
Mode of action
Good
Intermediate
Poor
Fungi
Antibiosis 'Mycoparasitism' Both Antibiosis Antibiosis
13 27 27 3 8
113 6 18 30 4
14 107 95 25 5
Bacteria Actinomycetes
A. M. Jackson, J. M. Whipps & J. M. Lynch
than good antibiotic producers but often strains exhibiting 'mycoparasitism' also had some antibiotic-producing capability. Most fungal isolates were poor mycoparasites or intermediate antibiotic producers.
Bacteria and actinomycetes. In volatile antipiotic tests,
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T score but the top 60 isolates in both PCA ranking and T score ranking were defined by near maximum scores for Form and PosBS. Subsequent PCA on these top 60 isolates gave a different ranking order to that obtained by PCA of all isolates and further indicated that within the top 30 isolates there was little discrimination in the T score system.
4 (6'9%) bacteria and 6 (35'3%) actinomycetes inhibited
mycelial growth of the pathogen by 25 % or more. Six (10'3 %) bacteria and 3 (17'6%) actinomycetes were fungitoxic to the pathogen and 14 (24'1 %) bacteria and 3 (17'6%) actinomycetes were fungistatic. The T scores of the bacteria ranged from 0 to 77 (Table 2). The four most active isolates (T scores of 67 to 77) were Pseudomonas or Alcaligenes spp. Of the 8 most active isolates, one had volatile antibiotic activity which inhibited mycelial growth by 41 %,6 produced fungitoxic non-volatile antibiotics and 2 produced fungistatic non-volatile antibiotics. T scores of actinomycetes ranged from 3 to 87. The highest-ranked isolate inhibited pathogen growth by 89% by the production of volatile antibiotics. Of the top 10 isolates with scores between 60 and 90, two produced fungistatic and three produced fungitoxic non-volatile antibiotics. In dual culture, pathogen growth was inhibited by 55 to 100%.
Multivariate analysis of the T score system To determine the relative contribution of assessments to the T score for fungi, a multivariate analysis using principal component analysis (PCA) was done (Morrison, 1967). Because the tests had different ranges, a correlation rather than a covariance matrix of the data was used. Negative values in the NVA. VA. DC and Form tests (see Table 1) were censored to zero. The multivariate analysis showed that T score was most highly correlated with PosBS and Form (Table 4) and the first principal component, which was essentially a combination of these two variates, accounted for 29% of the total variation. The first principal component score had a correlation of 0'882 with T score, 0'860 with PosBs and 0'825 with Form. The second principal component, which accounted for 24 % of the variation, was a simple mean of NVA. DC and Inter. Correlation of this second component with T score was poor as this component represented variation not accounted for by the first component which was closely related to T score. Overall the general ranking derived from PCA was similar to
Table 4. Major correlations (r) between T score and individual screening tests obtained using multivariate analysis Interactions' T score PosBS T score Inter T score PosBS NVA T score T score • See Table 1 for codes.
28
r value v. PosBS v. Form v. Form v.NVA v. Inter v. PosWS v. DC v. PosWS v. NVA
0'784 0'730 0'716 0'675 0'633 0'598 0'556 0'519 0'517
DISCUSSION The primary screening system included observations on the effects of micro-organisms on mycelial growth and sclerotia formation of Sclerotium cepivorum. It quantified isolate activity and provided some information on modes of action. Actinomycetes and bacteria produced both volatile and non-volatile antibiotics whereas the fungi only produced nonvolatile antibiotics that severely inhibited pathogen growth. Various effects on sclerotial formation were observed in response to test micro-organisms. Some isolates prevented the formation of sclerotial initials whereas others prevented their maturation, perhaps by blocking the metabolic pathway to pigment production. The availability of nutrients can influence sclerotial initial formation and sclerotia pigmentation (Townsend, 1957) and fast-growing isolates such as Trichoderma spp. may act in this way. Some fungi had no effect on sclerotial formation and maturation but in 40 % of cases the number of sclerotia was significantly lower than on control plates, indicating some more subtle interaction. Fungi, bacteria and actinomycetes have been tested before in dual-culture against S. cepivorum but an objective of this study was to design a screening system to enable modes of action to be distinguished as well as producing a general ranking of antagonism. For example, some Trichoderma isolates were highly effective in antibiosis whereas others were comparatively poor antibiotic producers. However, some of the latter invaded host colonies very rapidly, indicative of mycoparasitism which some previous screens may have neglected. However, statistical analyses of the T score system indicated that percent inhibition of sclerotia formation (Form) and position of black sclerotia formation (PosBS) formed the basic structure of the ranking, after which non-volatile antibiotic (NVA) and interaction type (Inter) assessments could be used for further discrimination. Nevertheless, these four assessments were not sensitive enough to differentiate the ranking in the top 30 isolates. So a cheaper and more efficient primary screen would have been achieved by considering just the initial two variates, Form and PosBS, and carrying out further screening on isolates with high scores in these alone. Interaction tests would be used in the next level of screening and would detect mycoparasitic potential. Indeed without interaction tests or VA tests (which were not discriminating in the T score system), 'mycoparasitism' and the potent volatile antibiotic produced by one of the actinomycetes would not have been detected. A multi-test screening system thus has advantages over a one- or two-test system if it represents several different modes of action. This approach to the selection of potentially useful antagonists for control of AWR may be of interest to plant pathologists in general. Importantly, when isolates with scores from throughout the T score range were tested subsequently in an in vivo MYC 95
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Screening for Allium white rot biocontrol agents bioassay of Entwistle & Spence (1988) there was a close association between the results of these in vitro and in vivo tests (A. R. Entwistle & J. Clarkson, in preparation).
We thank Drs A. R. Entwistle and R. T. Burchill (IHRWellesbourne) for their co-operation, John Fenlon and Andrew Mead for help with statistical analyses and experimental design and the Agricultural Genetics Company for financial support. We would also like to thank the many research workers throughout the world who provided fungal and bacterial isolates for use in this screen.
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