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Production of an antibiotic by Phoma exigua
Trans. Br. mycol. Soc. 55 (I), 67-75 (1970) Printed in Great Britain
PRODUCTION OF AN ANTIBIOTIC BY PHOMA EX/GUA By C. LOGAN
AND
R. O'NEILL
Faculty of Agriculture, The Queen's University, Belfast All isolates of Phoma exigua Desm. var. exigua and about 60% of isolates of P. exigua Desm. var. foueata (Foister) Boerema produce on 2% malt agar an antibiotic principle (Antibiotic' E ') which, in vitro, is antagonistic to a number of Gram-positive and Gram-negative bacteria and two fungal species. It is bactericidal and fungicidal. The compound was partially purified by paper chromatography and the active principle is considered to be a phenolic derivative. It is diffusible, water-soluble, heat-stable and has reducing properties. Isolates ofvar.Joveata which do not produce Antibiotic' E' differ consistently in cultural characteristics from those which do by the production of higher concentrations of anthraquinone pigments and more abundant aerial hyphae.
In Northern Ireland two varieties of Phoma exigua Desm. are associated with potato gangrene disease. Most gangrene infections are caused by P. exigua Desm. var. foveata (Foister) Boerema (P. foveata Foist.) whilst P. exigua var. exigua is occasionally associated with the disease (Logan, 1967). Boerema & Howeler (1967) found that all isolates of var. exigua but only some of var. foveata produce a colourless substance' E' which under alkaline conditions forms the pigments IX and p. Recently, Logan & Khan (1969) confirmed these findings and stated that substance 'E' has antibiotic properties. This paper describes the reaction of various bacteria and fungi to this antibiotic. MATERIALS AND METHODS
Phoma isolates Isolates of var. foveata, var. exigua and P. eupyrena were obtained from gangrene lesions on potato tubers of various cultivars grown in several districts in Northern Ireland. Details of the isolates, their reactions to the NH a and NaOH tests (Logan & Khan, 1969) and the methylene blue test (described below) are given in Table 1. Malt agar (2 % Allen and Hanbury's malt extract-t z % Oxoid agar no. 3; pH 4'5) was used for the isolation and the maintenance of Phoma isolates at 20 "C, The capacity of these isolates to produce the antibiotic principle was tested in the following media: malt agar, Czapek (Dox) agar, and Czapek (Dox) agar with glucose, malt, starch, fructose, maltose, salicin or mannitol substituted for sucrose. Bacterial isolates Details of these isolates are given in Table 2. Nos. 3 and 4 were received from Dr D. C. Graham, East Craigs, Scotland; nos. 17 and 18 from 5- 2
68
Transactions British Mycological Society
Dr J. E. Crosse, East MaIling, England; nos. 11-16 from the National Collection of Plant Pathogenic Bacteria and nos. 19-26 from the National Collection of Type Cultures. The Northern Ireland isolates were obtained from either soft-rotted potato tubers or black-leg infected plants using the citrate-pectate-gel selective medium (Logan, 1963). All isolates were maintained on nutrient agar (Oxoid CM3) at 26°. Table No. I
2 3 4 5 6 7 8 9 10 II 12
1.
Details
Isolate no. *
Identity
21067/3 81067/3B 101167/2 11067/3A 3 1067/5 7 1067/ 12 29 1167/1 5 1167/ 1 4 106715A 61067/2A 81067/3A 294/28
veicfoueata var·foveata var. foueata var. foueata vex.fooeata var. foveata var, foueata var. foueata var. exigua var. exigua P. eupyrena veacfooeata
lif Phoma isolates Antibiotic NHs test NaOHtest M.B. test production
+ + + + + + + +
+ + + +
+ + + +
+ + + +
+ +
+ +
+ +
+
+
+
+
* All isolates from
gangrene affected tubers grown in Northern Ireland except no. 294/28, which was received from Scotland.
Table 2. Details No. I
2 3 4 5 6 7 8 9 10 I I 12 13 1.4-
15 16 17 18 19 20 21 22 23 24 25 26
lif bacterial isolates
Isolate
Origin
Host
963/8 462/10 II9 V 14 B 362/23 1165/4 76 1/14 266/1 1065/1 I 365/1 9 25 1488 529 XL 59/9 1104 73 1 S20 CI5
N. Ireland N. Ireland Israel England N. Ireland N. Ireland N. Ireland N. Ireland N. Ireland N. Ireland S. Africa England England Uganda Hungary
Potato } Potato Tomato potato} Potato Potato potato} Potato Potato Potato Dahlia Sweet pea Cabbage Cotton French Bean Tobacco Pear Cherry
England England
607 1
59'" 801 5 )
8208 10375 657 8 800 7 860 3
N.C.T.C.
Name
Erwinia aroideae E. carotovora var. carotovora E. carotovora var. atroseptica Bacillus subtilis Agrobacterium tumefaciens Corynebacterium fascians Xanthomonas campestris X. malvacearum Pseudomonas phaseolicola P. tabaci P. syringae P. mors-prunorum Aerobacter cloacae Citrobacter freundii Serratia tnarcescens Shigella boydii Proteus morganii Hafnia alvei Escherichia coli E. coli
The reaction of these isolates to the antibiotic was tested in Petri dish cultures seeded by the poured-plate method, as follows. A bacterial suspension was prepared by adding 9 ml sterile Ringer's solution (quarter strength) to a 24-h-old nutrient agar slope culture and shaking vigorously.
Phoma exigua. C. Logan and R. O'Neill
69
A standard 0'02 mlloopful of the bacterial suspension was inoculated into 12'5 ml of Diagnostic Sensitivity Test Agar (Oxoid CM261) held at 41° and poured into 9 em Petri plates after thorough mixing. The antibiotic, usually in the form of a 5 mm diam block of malt agar and mycelium cut from a IO-day-old Phoma culture, was placed on the centre of the plate.
Fungal isolates Details of these and the medium on which spore production was induced are given in Table 3. Phytophthora infestans was maintained on raw potato slices, the other isolates on malt agar. Pycnidiospores of Phoma exigua varieties were produced on starch agar (Logan & Khan, 1969). Table 3. Details offungal isolates No.
Name
I
Fusarium caeruleum Phytophthora infestans Botrytis cinerea Aspergillus niger Penicillium expansum
2
3 4 5
Source Potato Potato Strawberry Contaminant Contaminant
Spore production on Malt agar Potato slices Strawberry fruit Malt agar Malt agar
The effect of the antibiotic on spore germination was tested by two methods. The first was in Petri dish cultures seeded by the poured plate method by adding 1 ml of a known concentration of spores (106 , 105 and 104 spores/ml) to 10 ml aliquots of malt agar. After the antibiotic had been applied to the cultures they were incubated at 20° for at least 4 days when the diameter of the inhibition zone was measured. The second method involved the observation of spore germination (105 sporesjml) in 1'0 % sucrose solution (Logan & Khan, 1969) after various dilutions of antibiotic had been added. The percentage of spores which germinated was assessed by microscopic examination of 100 spores in each of three separate drops from the various dilutions after 48 h incubation at 20°.
Demonstration of the antibiotic principle The simplest method of demonstrating whether or not an isolate of Phoma exigua produced the antibiotic principle was by transferring a 5 mm diam block of malt agar and mycelium from a r o-day-old culture to the surface of a Petri dish culture of E. aroideae (isolate 963/8) seeded by the poured plate method. If the isolate produced antibiotic, an inhibition zone of about 20 mm diam was clearly visible after 24 h incubation at 26°. A crude preparation of the antibiotic was prepared by soaking the agar plus mycelium of one 14-day-old malt agar culture from a 9 em Petri dish in 30 ml distilled water. After 24 h the agar and mycelium were removed by filtration. The filtrate was reduced by continuous boiling to about 0'25 ml and made up to 2 ml with Ringer's solution (quarter strength). The latter served as a standard stock solution from which dilution series were prepared. As controls, standard stock solutions were prepared in the same way from var. foveata isolates which did not produce the antibiotic.
70
Transactions British Mycological Society
The efficiency of various dilutions of the antibiotic in inhibiting bacterial growth was tested by filling 5 mm diam wells cut in the centre of poured plates. Methylene blue reduction test A stock solution was prepared by diluting 3 ml of 0'5 % (w/v) methylene blue in 200 ml distilled water. The test was carried out in a 75 x 8 mm test-tube to which 4-5 drops of stock solut.ion plus I drop N NaOH were added. The test material, in the form of either a 5 mm diam plug cut from a 7-10-day-old Phoma culture, or one drop of a crude or partially purified preparation, or a 10 mm square cut from a paper chromatogram, was added and the time taken to completely decolorize the methylene blue was noted. This test, which complements the NaOH test (Logan & Khan, 1969), was found necessary when examining crude or partially purified preparations for the presence of substance 'E'. For instance, when N NaOH is added to these preparations the result is an immediate red colour, the reaction being too rapid for the observation of t.he intermediate greenish blue pigment. Partial purification of the antibiotic A 25 ml aliquot of the crude preparation described above was deproteinized with acetone and the filtrate washed twice with ether and twice with chloroform to remove the anthraquinone pigments, the aqueous layer being retained on each occasion. The aqueous extract was then added to 400 ml industrial methylated spirit, the precipitate centrifuged down and the alcohol evaporated from the filtrate. At each stage the extracts, precipitates and filtrates were assayed for the presence of the ant.ibiotic against E. aroideae (isolate 963/8) and their reducing action also noted in methylene blue. After the alcohol was evaporated the substances remaining in the filtrate were separated by two-way paper chromatography, the first phase in butanol-acetic acid-water (4: I :5), the second in acetic acid (2 %, v/v). A reference chromatogram was developed in ferric chloride-ferricyanide reagent to detect phenolic compounds (Smith, 1960). These compounds were marked on the unstained chromatograms, the spots cut out, tested for the presence of the antibiotic principle by bio-assay against E. aroideae (isolate 963/8) and for their reducing ability in methylene blue. RESULTS
Cultural differences between'E' + ve and' E' - ve isolates of var. foveata Tests on the media listed above indicated that Phoma isolates produced the highest concentration of antibiotic in 2 % malt agar, the amount being slightly less in Czapek Dox agar with sucrose, glucose or malt as the carbohydrate source. No antibiotic was produced in Czapek Dox agar with salicin or mannitol substituted for sucrose, whilst variable results were obtained in this medium with starch, fructose or maltose substitutions. Antibiotic production in malt agar was detectable by bio-assay after
Phoma exigua. C. Logan and R. O'Neill
71
5 days incubation at 20°, the concentration being higher at the centre than at the edge of th e cultures. After 10 days, the concentration was uniform throughout the cultures and was even detectable in the agar medium 5-6 mm ahead of fungal growth. After testing several var. foveata isolates it became evident that they could be divided into two distinct groups, those which did and those which did not produce antibiotic. From a total of over 200 isolates from various districts in Northern Ireland it was found that 60 % of these produced the antibiotic principle. It was also found that, without exception, isolates of var. foveata which produced the antibiotic also produced substance ' E ' (Boerema & Howeler, 1967) and it was decided to name it , Antibiotic E'. Furthermore, all 'E' + ve isolates reduced methylene blue, a 5 mm diam agar plug causing decolorization in 3 min, whereas , E' - ve isolates caused no reaction (Table I). All var, exigua isolates tested produced the antibiotic principle, substance 'E' and also reduced methylene blue, a 5 mm diam agar plug causing decolourization in 5 min (Table I). With the discovery that isolates of var. foveata differed in their ability to produce an antibiotic principle, it became apparent th at there were also consistent cultural differences in the production of anthraquinone pigments (Bick & Rhee, 1966) and aerial hyphae. After 3 days incubation at 20° on 2 % malt agar a yellowish brown tint developed in the centre of isolates which did not produce the principle (' E ' - ve isolates ), whilst in ' E ' + ve isolates th e production of visible pigmentation was delayed until the 4th or 5th day. In addition, ' E' - ve isolates always became more highly pigmented than did ' E' + ve isolates. Also, ' E' - ve isolates produced abundant aerial hyphae which were scant or absent in 'E' + ve isolates. After 10 days growth, differences in pigmentation almost disappeared but the isolat es could still be characterized by the amount of aerial mycelial growth.
The effect of 'Antibiotic E ' on bacterial isolates The reaction of twenty-six bacterial isolates of extremely diverse origin to twelve Phoma isolates from potato are given in Table 4 as the diameter (mm) of the inhibition zone which developed 24 h after placing an agar plug on seeded agar plates. As previously stated, the var. foveata isolates formed two distinct groups, those which produced 'Antibiotic E' and those which did not. Isolate no. 12 (294/28 from Scotland) which forms abundant pycnidia in culture, produced less' Antibiotic E' than nos. 1-4 which rarely produce pycnidia in culture. It is also evident that var. exigua isolates produced less antibiotic than var. fo veata isolates 1-4. The isolate of P. eupyrena tested did not produce ' Antibiotic E '. The results in Table 4 show that ' Antibiotic E ' had a wide spectrum of activity against both Gram-positive (nos. 10-12 ) and the remaining Gram-negative bacterial isolates. It appeared particularly antagonistic to B. subtilis, the two Xanthomonas spp ., P. mors-prunorum, and least active against P. syringae. These tests were repeated with virtually identical inhibition zones.
-....]
N
Table 4. Diameter* of inhibition zones (mm) produced by bacterial isolates as a reaction to the agar block test Phoma isolates] A
No. I 2 3 4 S 6 7 8 9 10 II 12 13 14 IS 16 17 18 19 20 21 22 23 24 25 26
I
2
10
II
12
Isolate no.+
fOD. E+ve
fou. E+ve
fOD. E+ve
fOD. E+ve
fOD. E-ve
fou, E-ve
fou. E-ve
foo, E-ve
exig. E+ve
exig. E +ve
eup, E-ve
fOD. E+ve
9 63/8 4 6 2/ 10 II9 V 14 B 3 6 2/23 1165 / 4 7 6 1/ 14 266/1 1065/II 36S/1 9 25 14 8 8 5 29 XL59/9 11°4 73 1 S20 CI5 S9 2 0 60 7 1 801 5 8208 10 3 75 657 8 800 7 86°3
21 24 12 14 14 IS 14 16 20 28 IS 19 26 30 22 20 28 8 IS 12 14 23 18 19 18 18
19 22 II 13 14 12 14 14 20 26 IS IS 28 27 20 18 27 9 12 12 10 21 16 18 IS 16
21 23 13 14 14 13 IS 16 22 27 13 16 28
21 22 12 13 14 13 14 14 22 27 IS 17 28 28 19 18 28 8 16 12 13 22 19 19 14 17
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
10 12 0 0 0 0 0 0
II 14 0 0 0 0 0 0 0 14 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
13 16
3
4
S
6
7
8
9
~
$::l
*
3° 24 19 28 8 14 12 14 23 17 19 16 19
Mean of four replicates.
0
0 0 0 0 0 0 0 0 0 0
0
0
0 0 0 0
0
0
0
0
0 0 0
t
0
Details in Table I.
0
0 0 0 0 0
0
0 0 0 0
0 0 0 0 0 0 0
0 0 0
0
+ Details in Table 2.
0
12 0 0 0
7 0 10
I7 0 7 0 0 9 7 7 0 0
0
0 7 0 12 21 0 9 0 0 II 7 9 0 °
0 0 0
0 0
7 9 9 8 0 0 12 19 7 0
19 12 7 II 14 7 10 9 9 16 II 14 II
II
~
~ ....
~.
b:l
....
;:to
~
~ (") ~
~ o-q
~.
(::j .......
~
(")
~.
~
73
Phoma exigua. C. Logan and R. O'Neill
Table 5 shows the results of the reaction of E. aroideae (isolate 963/8) to various dilutions of a crude preparation of 'Antibiotic E' from Phoma isolate 101167/2 and the reducing power of the dilutions on methylene blue. These indicate that the antibiotic could be concentrated by boiling without loss of activity and was still reasonably active at a dilution of 1 : 64. The crude preparation of the 'E' -ve isolate ofvar.foveata (71067/12) had no antibiotic properties nor did it reduce methylene blue. When 5 mm diam blocks of agar were removed from within the inhibition zone to nutrient broth and incubated at 26° for 1 week no bacterial growth occurred, indicating that' Antibiotic E' was bactericidal. Table 5. The diameter of inhibition zones and methylene blue reaction produced by dilutions of crude preparations of' E' + ve and' E' - ve var. foveata isolates Diameter* (mm) test bacterium 963/8
Time (min) to reduce methylene blue
"
A
Dilution
E+ve
E-ve
I: I 1:2 1:4 1:8 1:16 1:32 1:64 1:128
35 30 25 21 21 16 15 0
o
I
o o
2
E+ve
3
o
10 12
o o o o
* = Mean of four replicates.
-
E-ve
= No reduction of methylene blue.
Table 6. The effect of dilutions of crude preparations of' E' + ve and' E' - ve isolates on the percentage germination ofspores (ex 300) of Fusarium caeruleum and Phytophthora infestans Percentage germination
,
F. caeruleum
P. infestans
Dilution
E+ve
E-ve
I: I 1:2 1:4 1:8 I: 16 1:3 2
0 0 48 92 100 100
100 100 100 100 100 100
E+ve 0 0 0 0 0 10
E-ve 0 64 78 85 83 85
The effect of' Antibiotic E' onfungal isolates The effect on the germination of Fusarium caeruleum and Phytophthora infestans spores in a 1 % sucrose solution when various dilutions of crude preparations of 'E' + ve and 'E' - ve var. foveata isolates were added is given in Table 6. The same dilutions did not affect the germination of spores of the other fungi tested, namely P. exigua var. exigua, P. exigua var. foveata, Botrytis cinerea, Aspergillus niger and Penicillium expansum. When the effect of 'Antibiotic E' on the germination of spores of F. caeruleum, P. expansum and A. niger was tested by the poured plate method, inhibition zones were apparent only in plates seeded with F. caeruleum spores. The diameter of the zone varied inversely with spore concentration.
74
Transactions British Mycological Society
For instance, plates seeded with 106, 105 and 104 spores/ml of F. caeruleum produced zones of diameter 24, 36 and 45 mm respectively. The effect of the antibiotic on the germination of F. caeruleum spores in 1 % sucrose was different from that in malt agar. In the sucrose solution the spores either germinated normally or did not germinate at all. In seeded malt agar plates the spores within the inhibition zone produced germ-tubes, the tips of which became swollen and eventually lysisoccurred.
Partial purification oj the antibiotic I t was found that most of the antibiotic principle remained in the filtrate following precipitation with industrial methylated spirits. The diameters of the inhibition zones in poured plates of E. aroideae (963/8) caused by the crude preparation, the alcoholic precipitate and the alcoholic filtrate were 25, 12 and 35 mm respectively. The crude preparation reduced methylene blue in 2 min, the alcoholic precipitate gave no reaction whilst the alcoholic filtrate decolourized it instantly. When the chromatograms were stained in ferric chloride-ferricyanide reagent, three spots gave a positive reaction (intense blue stain). One of these, whose RF value in the butanol-acetic acid-water phase was 0·88 and in the acetic acid phase was 0,86, produced an 18-20 mm inhibition zone in poured plates of E. aroideae (963/8) and also decolorized methylene blue within 3 min. Elutes from the other two spots possessed no antibiotic or reducing activity. Elutes from the spot which contained' Antibiotic E' retained their antibiotic properties after boiling for 5 min. DISCUSSION
It is now clear that within the species Phoma exigua three groups may be differentiated, (I) P. exigua var. exigua, (2) P. exigua var.joveata ('E' +ve) and (3) P. exigua var. joveata ('E' -ve). The latter two are readily distinguished from var. exigua by the cultural and pathogenic differences listed by Logan & Khan (1969), the ammonia test being particularly useful in demonstrating the presence of anthraquinone pigments produced by the two groups of var. joveata. Isolates within var. fooeata may also be distinguished by their cultural characteristics but more reliably by bioassay for antibiotic production, by the NaOH test for substance 'E' or by the methylene blue test. Pathogenicity studies have shown that the two groups ofvar.joveata differ slightly in virulence and produce different types of rot in potato tubers (C. Logan, unpublished). In the most recent review of antibiotics produced by fungi, Broadbent (1966) mentions only one species of Phoma, P. longissima which is said to have fungicidal properties (Voros, 1958). The discovery that P. exigua varieties produce an antibiotic was not purely by chance but was initiated by the observation of two phenomena that have occurred in potato tuberborne diseases during recent years. The first happens with the disease-condition known as skin necrosis, with which the two organisms P. exigua var. joveata and E. carotovora var. atroseptica are often found associated (Todd, 1963). From these shallow
Phoma exigua. C. Logan and R. O'Neill
75
lesions the fungus is more commonly isolated than the bacterium whereas under laboratory conditions the symptoms of skin necrosis are readily reproduced by the bacterium (Logan, 1964) but not by the fungus. The second is the fact that from the time potato gangrene was distinguished from dry rot (Alcock & Foister, 1936) its incidence has gradually increased whereas, by contrast, dry rot has become less important as a storage rot of seed potato tubers. It is reasonable to infer that in a narrow zone of competition between various micro-organisms, antibiotic production may be of biological significance in the colonization of a substrate (Garrett, 1956), in this case the damaged surface of the potato tuber might well provide sufficient nutrients for significant antibiotic production. Hence infection experiments with mixed inocula of the pathogens concerned are now being carried out to find out to what extent, if any, 'Antibiotic E' enhances infection by var. foveata over its competitors on tuber surfaces. REFERENCES
ALCOCK, N. L. & FOISTER, C. E. (1936). A fungus disease of stored potatoes. Scott. ]. Agric. 19, 252-257. BICK, I. R. C. & RHEE, C. (1966). Anthraquinone pigments from Phomafoveata Foister. Biochem. ].98, 112-II6. BOEREMA, G. H. & H6wELER, L. H. (1967). Phoma exigua Desm. and its varieties. Persoonia 5, 1-12. BROADBENT, D. (1966). Antibiotics produced by fungi. Bot. Rev. 32, 219-242. GARRETT, S. D. (1956). Biology of root-infecting fungi. Cambridge University Press. LOGAN, C. (1963). A selective medium for the isolation of soft rot coliforms from soil. Nature, Lond. 199, 623. LOGAN, C. (1964). Bacterial hard rot of potato. Eur. Potato ]. 7, 45-56. LOGAN, C. (1967). Potato stem infection by Phoma solanicola Prill. & Delacr. f. foveata (Foister) Malcolmson. Pl. Path. 16, 64-67. LOGAN, C. & KHAN, A. A. (1969)' Comparative studies of Phoma spp. associated with potato gangrene in Northern Ireland. Trans. Br. mycol. Soc. 52. 9-17. SMITH, I. (1960). Chromatographic and Electrophoretic techniques. Vol. I, Chromatography. London: W. Heinemann Ltd. TODD, J. M. (1963). The development and control of potato dry rot, gangrene and skin spot. SeedPotato 3, 14-18. VOROS, J. (1958). Fungistatic activity of the Spaeropsidales and Melanconiales. Acta microbiol. 5, 261-266.
(Acceptedfor publication
10
February 1970)
PRODUCTION OF AN ANTIBIOTIC BY PHOMA EX/GUA By C. LOGAN
AND
R. O'NEILL
Faculty of Agriculture, The Queen's University, Belfast All isolates of Phoma exigua Desm. var. exigua and about 60% of isolates of P. exigua Desm. var. foueata (Foister) Boerema produce on 2% malt agar an antibiotic principle (Antibiotic' E ') which, in vitro, is antagonistic to a number of Gram-positive and Gram-negative bacteria and two fungal species. It is bactericidal and fungicidal. The compound was partially purified by paper chromatography and the active principle is considered to be a phenolic derivative. It is diffusible, water-soluble, heat-stable and has reducing properties. Isolates ofvar.Joveata which do not produce Antibiotic' E' differ consistently in cultural characteristics from those which do by the production of higher concentrations of anthraquinone pigments and more abundant aerial hyphae.
In Northern Ireland two varieties of Phoma exigua Desm. are associated with potato gangrene disease. Most gangrene infections are caused by P. exigua Desm. var. foveata (Foister) Boerema (P. foveata Foist.) whilst P. exigua var. exigua is occasionally associated with the disease (Logan, 1967). Boerema & Howeler (1967) found that all isolates of var. exigua but only some of var. foveata produce a colourless substance' E' which under alkaline conditions forms the pigments IX and p. Recently, Logan & Khan (1969) confirmed these findings and stated that substance 'E' has antibiotic properties. This paper describes the reaction of various bacteria and fungi to this antibiotic. MATERIALS AND METHODS
Phoma isolates Isolates of var. foveata, var. exigua and P. eupyrena were obtained from gangrene lesions on potato tubers of various cultivars grown in several districts in Northern Ireland. Details of the isolates, their reactions to the NH a and NaOH tests (Logan & Khan, 1969) and the methylene blue test (described below) are given in Table 1. Malt agar (2 % Allen and Hanbury's malt extract-t z % Oxoid agar no. 3; pH 4'5) was used for the isolation and the maintenance of Phoma isolates at 20 "C, The capacity of these isolates to produce the antibiotic principle was tested in the following media: malt agar, Czapek (Dox) agar, and Czapek (Dox) agar with glucose, malt, starch, fructose, maltose, salicin or mannitol substituted for sucrose. Bacterial isolates Details of these isolates are given in Table 2. Nos. 3 and 4 were received from Dr D. C. Graham, East Craigs, Scotland; nos. 17 and 18 from 5- 2
68
Transactions British Mycological Society
Dr J. E. Crosse, East MaIling, England; nos. 11-16 from the National Collection of Plant Pathogenic Bacteria and nos. 19-26 from the National Collection of Type Cultures. The Northern Ireland isolates were obtained from either soft-rotted potato tubers or black-leg infected plants using the citrate-pectate-gel selective medium (Logan, 1963). All isolates were maintained on nutrient agar (Oxoid CM3) at 26°. Table No. I
2 3 4 5 6 7 8 9 10 II 12
1.
Details
Isolate no. *
Identity
21067/3 81067/3B 101167/2 11067/3A 3 1067/5 7 1067/ 12 29 1167/1 5 1167/ 1 4 106715A 61067/2A 81067/3A 294/28
veicfoueata var·foveata var. foueata var. foueata vex.fooeata var. foveata var, foueata var. foueata var. exigua var. exigua P. eupyrena veacfooeata
lif Phoma isolates Antibiotic NHs test NaOHtest M.B. test production
+ + + + + + + +
+ + + +
+ + + +
+ + + +
+ +
+ +
+ +
+
+
+
+
* All isolates from
gangrene affected tubers grown in Northern Ireland except no. 294/28, which was received from Scotland.
Table 2. Details No. I
2 3 4 5 6 7 8 9 10 I I 12 13 1.4-
15 16 17 18 19 20 21 22 23 24 25 26
lif bacterial isolates
Isolate
Origin
Host
963/8 462/10 II9 V 14 B 362/23 1165/4 76 1/14 266/1 1065/1 I 365/1 9 25 1488 529 XL 59/9 1104 73 1 S20 CI5
N. Ireland N. Ireland Israel England N. Ireland N. Ireland N. Ireland N. Ireland N. Ireland N. Ireland S. Africa England England Uganda Hungary
Potato } Potato Tomato potato} Potato Potato potato} Potato Potato Potato Dahlia Sweet pea Cabbage Cotton French Bean Tobacco Pear Cherry
England England
607 1
59'" 801 5 )
8208 10375 657 8 800 7 860 3
N.C.T.C.
Name
Erwinia aroideae E. carotovora var. carotovora E. carotovora var. atroseptica Bacillus subtilis Agrobacterium tumefaciens Corynebacterium fascians Xanthomonas campestris X. malvacearum Pseudomonas phaseolicola P. tabaci P. syringae P. mors-prunorum Aerobacter cloacae Citrobacter freundii Serratia tnarcescens Shigella boydii Proteus morganii Hafnia alvei Escherichia coli E. coli
The reaction of these isolates to the antibiotic was tested in Petri dish cultures seeded by the poured-plate method, as follows. A bacterial suspension was prepared by adding 9 ml sterile Ringer's solution (quarter strength) to a 24-h-old nutrient agar slope culture and shaking vigorously.
Phoma exigua. C. Logan and R. O'Neill
69
A standard 0'02 mlloopful of the bacterial suspension was inoculated into 12'5 ml of Diagnostic Sensitivity Test Agar (Oxoid CM261) held at 41° and poured into 9 em Petri plates after thorough mixing. The antibiotic, usually in the form of a 5 mm diam block of malt agar and mycelium cut from a IO-day-old Phoma culture, was placed on the centre of the plate.
Fungal isolates Details of these and the medium on which spore production was induced are given in Table 3. Phytophthora infestans was maintained on raw potato slices, the other isolates on malt agar. Pycnidiospores of Phoma exigua varieties were produced on starch agar (Logan & Khan, 1969). Table 3. Details offungal isolates No.
Name
I
Fusarium caeruleum Phytophthora infestans Botrytis cinerea Aspergillus niger Penicillium expansum
2
3 4 5
Source Potato Potato Strawberry Contaminant Contaminant
Spore production on Malt agar Potato slices Strawberry fruit Malt agar Malt agar
The effect of the antibiotic on spore germination was tested by two methods. The first was in Petri dish cultures seeded by the poured plate method by adding 1 ml of a known concentration of spores (106 , 105 and 104 spores/ml) to 10 ml aliquots of malt agar. After the antibiotic had been applied to the cultures they were incubated at 20° for at least 4 days when the diameter of the inhibition zone was measured. The second method involved the observation of spore germination (105 sporesjml) in 1'0 % sucrose solution (Logan & Khan, 1969) after various dilutions of antibiotic had been added. The percentage of spores which germinated was assessed by microscopic examination of 100 spores in each of three separate drops from the various dilutions after 48 h incubation at 20°.
Demonstration of the antibiotic principle The simplest method of demonstrating whether or not an isolate of Phoma exigua produced the antibiotic principle was by transferring a 5 mm diam block of malt agar and mycelium from a r o-day-old culture to the surface of a Petri dish culture of E. aroideae (isolate 963/8) seeded by the poured plate method. If the isolate produced antibiotic, an inhibition zone of about 20 mm diam was clearly visible after 24 h incubation at 26°. A crude preparation of the antibiotic was prepared by soaking the agar plus mycelium of one 14-day-old malt agar culture from a 9 em Petri dish in 30 ml distilled water. After 24 h the agar and mycelium were removed by filtration. The filtrate was reduced by continuous boiling to about 0'25 ml and made up to 2 ml with Ringer's solution (quarter strength). The latter served as a standard stock solution from which dilution series were prepared. As controls, standard stock solutions were prepared in the same way from var. foveata isolates which did not produce the antibiotic.
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Transactions British Mycological Society
The efficiency of various dilutions of the antibiotic in inhibiting bacterial growth was tested by filling 5 mm diam wells cut in the centre of poured plates. Methylene blue reduction test A stock solution was prepared by diluting 3 ml of 0'5 % (w/v) methylene blue in 200 ml distilled water. The test was carried out in a 75 x 8 mm test-tube to which 4-5 drops of stock solut.ion plus I drop N NaOH were added. The test material, in the form of either a 5 mm diam plug cut from a 7-10-day-old Phoma culture, or one drop of a crude or partially purified preparation, or a 10 mm square cut from a paper chromatogram, was added and the time taken to completely decolorize the methylene blue was noted. This test, which complements the NaOH test (Logan & Khan, 1969), was found necessary when examining crude or partially purified preparations for the presence of substance 'E'. For instance, when N NaOH is added to these preparations the result is an immediate red colour, the reaction being too rapid for the observation of t.he intermediate greenish blue pigment. Partial purification of the antibiotic A 25 ml aliquot of the crude preparation described above was deproteinized with acetone and the filtrate washed twice with ether and twice with chloroform to remove the anthraquinone pigments, the aqueous layer being retained on each occasion. The aqueous extract was then added to 400 ml industrial methylated spirit, the precipitate centrifuged down and the alcohol evaporated from the filtrate. At each stage the extracts, precipitates and filtrates were assayed for the presence of the ant.ibiotic against E. aroideae (isolate 963/8) and their reducing action also noted in methylene blue. After the alcohol was evaporated the substances remaining in the filtrate were separated by two-way paper chromatography, the first phase in butanol-acetic acid-water (4: I :5), the second in acetic acid (2 %, v/v). A reference chromatogram was developed in ferric chloride-ferricyanide reagent to detect phenolic compounds (Smith, 1960). These compounds were marked on the unstained chromatograms, the spots cut out, tested for the presence of the antibiotic principle by bio-assay against E. aroideae (isolate 963/8) and for their reducing ability in methylene blue. RESULTS
Cultural differences between'E' + ve and' E' - ve isolates of var. foveata Tests on the media listed above indicated that Phoma isolates produced the highest concentration of antibiotic in 2 % malt agar, the amount being slightly less in Czapek Dox agar with sucrose, glucose or malt as the carbohydrate source. No antibiotic was produced in Czapek Dox agar with salicin or mannitol substituted for sucrose, whilst variable results were obtained in this medium with starch, fructose or maltose substitutions. Antibiotic production in malt agar was detectable by bio-assay after
Phoma exigua. C. Logan and R. O'Neill
71
5 days incubation at 20°, the concentration being higher at the centre than at the edge of th e cultures. After 10 days, the concentration was uniform throughout the cultures and was even detectable in the agar medium 5-6 mm ahead of fungal growth. After testing several var. foveata isolates it became evident that they could be divided into two distinct groups, those which did and those which did not produce antibiotic. From a total of over 200 isolates from various districts in Northern Ireland it was found that 60 % of these produced the antibiotic principle. It was also found that, without exception, isolates of var. foveata which produced the antibiotic also produced substance ' E ' (Boerema & Howeler, 1967) and it was decided to name it , Antibiotic E'. Furthermore, all 'E' + ve isolates reduced methylene blue, a 5 mm diam agar plug causing decolorization in 3 min, whereas , E' - ve isolates caused no reaction (Table I). All var, exigua isolates tested produced the antibiotic principle, substance 'E' and also reduced methylene blue, a 5 mm diam agar plug causing decolourization in 5 min (Table I). With the discovery that isolates of var. foveata differed in their ability to produce an antibiotic principle, it became apparent th at there were also consistent cultural differences in the production of anthraquinone pigments (Bick & Rhee, 1966) and aerial hyphae. After 3 days incubation at 20° on 2 % malt agar a yellowish brown tint developed in the centre of isolates which did not produce the principle (' E ' - ve isolates ), whilst in ' E ' + ve isolates th e production of visible pigmentation was delayed until the 4th or 5th day. In addition, ' E' - ve isolates always became more highly pigmented than did ' E' + ve isolates. Also, ' E' - ve isolates produced abundant aerial hyphae which were scant or absent in 'E' + ve isolates. After 10 days growth, differences in pigmentation almost disappeared but the isolat es could still be characterized by the amount of aerial mycelial growth.
The effect of 'Antibiotic E ' on bacterial isolates The reaction of twenty-six bacterial isolates of extremely diverse origin to twelve Phoma isolates from potato are given in Table 4 as the diameter (mm) of the inhibition zone which developed 24 h after placing an agar plug on seeded agar plates. As previously stated, the var. foveata isolates formed two distinct groups, those which produced 'Antibiotic E' and those which did not. Isolate no. 12 (294/28 from Scotland) which forms abundant pycnidia in culture, produced less' Antibiotic E' than nos. 1-4 which rarely produce pycnidia in culture. It is also evident that var. exigua isolates produced less antibiotic than var. fo veata isolates 1-4. The isolate of P. eupyrena tested did not produce ' Antibiotic E '. The results in Table 4 show that ' Antibiotic E ' had a wide spectrum of activity against both Gram-positive (nos. 10-12 ) and the remaining Gram-negative bacterial isolates. It appeared particularly antagonistic to B. subtilis, the two Xanthomonas spp ., P. mors-prunorum, and least active against P. syringae. These tests were repeated with virtually identical inhibition zones.
-....]
N
Table 4. Diameter* of inhibition zones (mm) produced by bacterial isolates as a reaction to the agar block test Phoma isolates] A
No. I 2 3 4 S 6 7 8 9 10 II 12 13 14 IS 16 17 18 19 20 21 22 23 24 25 26
I
2
10
II
12
Isolate no.+
fOD. E+ve
fou. E+ve
fOD. E+ve
fOD. E+ve
fOD. E-ve
fou, E-ve
fou. E-ve
foo, E-ve
exig. E+ve
exig. E +ve
eup, E-ve
fOD. E+ve
9 63/8 4 6 2/ 10 II9 V 14 B 3 6 2/23 1165 / 4 7 6 1/ 14 266/1 1065/II 36S/1 9 25 14 8 8 5 29 XL59/9 11°4 73 1 S20 CI5 S9 2 0 60 7 1 801 5 8208 10 3 75 657 8 800 7 86°3
21 24 12 14 14 IS 14 16 20 28 IS 19 26 30 22 20 28 8 IS 12 14 23 18 19 18 18
19 22 II 13 14 12 14 14 20 26 IS IS 28 27 20 18 27 9 12 12 10 21 16 18 IS 16
21 23 13 14 14 13 IS 16 22 27 13 16 28
21 22 12 13 14 13 14 14 22 27 IS 17 28 28 19 18 28 8 16 12 13 22 19 19 14 17
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
10 12 0 0 0 0 0 0
II 14 0 0 0 0 0 0 0 14 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
13 16
3
4
S
6
7
8
9
~
$::l
*
3° 24 19 28 8 14 12 14 23 17 19 16 19
Mean of four replicates.
0
0 0 0 0 0 0 0 0 0 0
0
0
0 0 0 0
0
0
0
0
0 0 0
t
0
Details in Table I.
0
0 0 0 0 0
0
0 0 0 0
0 0 0 0 0 0 0
0 0 0
0
+ Details in Table 2.
0
12 0 0 0
7 0 10
I7 0 7 0 0 9 7 7 0 0
0
0 7 0 12 21 0 9 0 0 II 7 9 0 °
0 0 0
0 0
7 9 9 8 0 0 12 19 7 0
19 12 7 II 14 7 10 9 9 16 II 14 II
II
~
~ ....
~.
b:l
....
;:to
~
~ (") ~
~ o-q
~.
(::j .......
~
(")
~.
~
73
Phoma exigua. C. Logan and R. O'Neill
Table 5 shows the results of the reaction of E. aroideae (isolate 963/8) to various dilutions of a crude preparation of 'Antibiotic E' from Phoma isolate 101167/2 and the reducing power of the dilutions on methylene blue. These indicate that the antibiotic could be concentrated by boiling without loss of activity and was still reasonably active at a dilution of 1 : 64. The crude preparation of the 'E' -ve isolate ofvar.foveata (71067/12) had no antibiotic properties nor did it reduce methylene blue. When 5 mm diam blocks of agar were removed from within the inhibition zone to nutrient broth and incubated at 26° for 1 week no bacterial growth occurred, indicating that' Antibiotic E' was bactericidal. Table 5. The diameter of inhibition zones and methylene blue reaction produced by dilutions of crude preparations of' E' + ve and' E' - ve var. foveata isolates Diameter* (mm) test bacterium 963/8
Time (min) to reduce methylene blue
"
A
Dilution
E+ve
E-ve
I: I 1:2 1:4 1:8 1:16 1:32 1:64 1:128
35 30 25 21 21 16 15 0
o
I
o o
2
E+ve
3
o
10 12
o o o o
* = Mean of four replicates.
-
E-ve
= No reduction of methylene blue.
Table 6. The effect of dilutions of crude preparations of' E' + ve and' E' - ve isolates on the percentage germination ofspores (ex 300) of Fusarium caeruleum and Phytophthora infestans Percentage germination
,
F. caeruleum
P. infestans
Dilution
E+ve
E-ve
I: I 1:2 1:4 1:8 I: 16 1:3 2
0 0 48 92 100 100
100 100 100 100 100 100
E+ve 0 0 0 0 0 10
E-ve 0 64 78 85 83 85
The effect of' Antibiotic E' onfungal isolates The effect on the germination of Fusarium caeruleum and Phytophthora infestans spores in a 1 % sucrose solution when various dilutions of crude preparations of 'E' + ve and 'E' - ve var. foveata isolates were added is given in Table 6. The same dilutions did not affect the germination of spores of the other fungi tested, namely P. exigua var. exigua, P. exigua var. foveata, Botrytis cinerea, Aspergillus niger and Penicillium expansum. When the effect of 'Antibiotic E' on the germination of spores of F. caeruleum, P. expansum and A. niger was tested by the poured plate method, inhibition zones were apparent only in plates seeded with F. caeruleum spores. The diameter of the zone varied inversely with spore concentration.
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Transactions British Mycological Society
For instance, plates seeded with 106, 105 and 104 spores/ml of F. caeruleum produced zones of diameter 24, 36 and 45 mm respectively. The effect of the antibiotic on the germination of F. caeruleum spores in 1 % sucrose was different from that in malt agar. In the sucrose solution the spores either germinated normally or did not germinate at all. In seeded malt agar plates the spores within the inhibition zone produced germ-tubes, the tips of which became swollen and eventually lysisoccurred.
Partial purification oj the antibiotic I t was found that most of the antibiotic principle remained in the filtrate following precipitation with industrial methylated spirits. The diameters of the inhibition zones in poured plates of E. aroideae (963/8) caused by the crude preparation, the alcoholic precipitate and the alcoholic filtrate were 25, 12 and 35 mm respectively. The crude preparation reduced methylene blue in 2 min, the alcoholic precipitate gave no reaction whilst the alcoholic filtrate decolourized it instantly. When the chromatograms were stained in ferric chloride-ferricyanide reagent, three spots gave a positive reaction (intense blue stain). One of these, whose RF value in the butanol-acetic acid-water phase was 0·88 and in the acetic acid phase was 0,86, produced an 18-20 mm inhibition zone in poured plates of E. aroideae (963/8) and also decolorized methylene blue within 3 min. Elutes from the other two spots possessed no antibiotic or reducing activity. Elutes from the spot which contained' Antibiotic E' retained their antibiotic properties after boiling for 5 min. DISCUSSION
It is now clear that within the species Phoma exigua three groups may be differentiated, (I) P. exigua var. exigua, (2) P. exigua var.joveata ('E' +ve) and (3) P. exigua var. joveata ('E' -ve). The latter two are readily distinguished from var. exigua by the cultural and pathogenic differences listed by Logan & Khan (1969), the ammonia test being particularly useful in demonstrating the presence of anthraquinone pigments produced by the two groups of var. joveata. Isolates within var. fooeata may also be distinguished by their cultural characteristics but more reliably by bioassay for antibiotic production, by the NaOH test for substance 'E' or by the methylene blue test. Pathogenicity studies have shown that the two groups ofvar.joveata differ slightly in virulence and produce different types of rot in potato tubers (C. Logan, unpublished). In the most recent review of antibiotics produced by fungi, Broadbent (1966) mentions only one species of Phoma, P. longissima which is said to have fungicidal properties (Voros, 1958). The discovery that P. exigua varieties produce an antibiotic was not purely by chance but was initiated by the observation of two phenomena that have occurred in potato tuberborne diseases during recent years. The first happens with the disease-condition known as skin necrosis, with which the two organisms P. exigua var. joveata and E. carotovora var. atroseptica are often found associated (Todd, 1963). From these shallow
Phoma exigua. C. Logan and R. O'Neill
75
lesions the fungus is more commonly isolated than the bacterium whereas under laboratory conditions the symptoms of skin necrosis are readily reproduced by the bacterium (Logan, 1964) but not by the fungus. The second is the fact that from the time potato gangrene was distinguished from dry rot (Alcock & Foister, 1936) its incidence has gradually increased whereas, by contrast, dry rot has become less important as a storage rot of seed potato tubers. It is reasonable to infer that in a narrow zone of competition between various micro-organisms, antibiotic production may be of biological significance in the colonization of a substrate (Garrett, 1956), in this case the damaged surface of the potato tuber might well provide sufficient nutrients for significant antibiotic production. Hence infection experiments with mixed inocula of the pathogens concerned are now being carried out to find out to what extent, if any, 'Antibiotic E' enhances infection by var. foveata over its competitors on tuber surfaces. REFERENCES
ALCOCK, N. L. & FOISTER, C. E. (1936). A fungus disease of stored potatoes. Scott. ]. Agric. 19, 252-257. BICK, I. R. C. & RHEE, C. (1966). Anthraquinone pigments from Phomafoveata Foister. Biochem. ].98, 112-II6. BOEREMA, G. H. & H6wELER, L. H. (1967). Phoma exigua Desm. and its varieties. Persoonia 5, 1-12. BROADBENT, D. (1966). Antibiotics produced by fungi. Bot. Rev. 32, 219-242. GARRETT, S. D. (1956). Biology of root-infecting fungi. Cambridge University Press. LOGAN, C. (1963). A selective medium for the isolation of soft rot coliforms from soil. Nature, Lond. 199, 623. LOGAN, C. (1964). Bacterial hard rot of potato. Eur. Potato ]. 7, 45-56. LOGAN, C. (1967). Potato stem infection by Phoma solanicola Prill. & Delacr. f. foveata (Foister) Malcolmson. Pl. Path. 16, 64-67. LOGAN, C. & KHAN, A. A. (1969)' Comparative studies of Phoma spp. associated with potato gangrene in Northern Ireland. Trans. Br. mycol. Soc. 52. 9-17. SMITH, I. (1960). Chromatographic and Electrophoretic techniques. Vol. I, Chromatography. London: W. Heinemann Ltd. TODD, J. M. (1963). The development and control of potato dry rot, gangrene and skin spot. SeedPotato 3, 14-18. VOROS, J. (1958). Fungistatic activity of the Spaeropsidales and Melanconiales. Acta microbiol. 5, 261-266.
(Acceptedfor publication
10
February 1970)