Trans. Br. mycol. Soc. 73 (1) 91-97 (1979) Printed in Great Britain

FACTORS AFFECTING SURVIVAL OF PHOMA EXIGUAVAR. FOVEATA IN SOIL

By M. J. ADAMS

Rothamsted Experimental Station, Harpenden, Herts., AL5 2JQ

The effects of temperature, soil moisture, soil type and fungal isolate on the longevity ofPhoma exigua Desm. var. foueata (Foister) Boerema (the cause of potato gangrene) in soilwere investigated by inoculating soils with pycnidiospores and incubating under constantconditions of temperature and moisture. There was an immediate, steady decline in fungalpopulations in all experiments, except in autoclaved soil where they initially remained con-stant for 3-4 months. Survival was prolonged by lower temperatures, populations in loambecoming undetectable within about 8 months at 15 DC, 2 years at 100 and 3 years at 50. Ingeneral survival was better (i.e. populations were larger at any sample time) the drier the soil,but in very dry conditions « 20 % water holding capacity) it was as poor as in soils at ornear field capacity. The fungus survived equally well in loam, clay, sandy-loam and peatsoils at the same moisture contents, measured as percentage water holding capacity. Onefungal isolate, which had been maintained in culture for several years, survived poorly butthere were no consistent differences between five other isolates.

The implication of these results is discussed in relation to survival in the field. Thesoil is unlikely to be an important reservoir of inoculum for potato crops, except possiblyin the production of high-grade seed stocks, where even small populations could resultin introduction of disease.

Phoma exigua Desm. is a ubiquitous weak patho-gen that has been isolated from a wide range ofhost plants and is assumed to be soil-borne(Boerema & Howeler, 1967). The var. fooeata(Foister) Boerema is known to produce symptomsonly on potato and Chenopodium (Boerema, 1977)and is the predominant cause in Western Europeof potato gangrene, which is currently one of themore important potato storage diseases in theUnited Kingdom. The fungus produces pycnidiaon senescing stems and a rot on tubers whichusually begins when infested soil is introducedinto wounds made at harvest or during subsequenthandling. The main source of inoculum for stemsand progeny tubers is probably seed tubers thatare rotting or contaminated with infested soil atplanting (Logan, 1974) but the importance of soilas a reservoir of inoculum is uncertain.

In Northern Ireland, Logan (1974) found thatwhen potatoes were grown in a rotation once every5 years, soil inoculum was reduced to a very lowlevel between potato crops and, in fields at Roth-amsted where potatoes are grown once every 7years, the pathogen has never been detected insoil sampled just before planting a potato crop.However, there are reports of the fungus survivingin soil for up to 5 years in Northern Ireland (Khan& Logan, 1968; Anon., 1977) and 7 years in northeast Scotland (Malcolmson & Gray, 1968). If

inoculum survived in air-dry soils on equipmentor in stores, this could also be a source of tubercontamination, especially in the production of highgrade disease-free seed stocks (Hide, 1978).

When soil has been inoculated with the fungus,its population has declined rapidly (Todd &Adam,1967; Entwistle, 1971; Fox, Dashwood & Wilson,1976, 1977) but the influence of environmentalconditions on survival is not known. This paperdescribes the results of experiments designed toassess the effects of temperature, soil moisture andsoil type on the survival of P. exigua var. fooeata.

MATERIALS AND METHODS

Soil inoculation

The four soils used in the experiments were (a)Kettering loam of pH 6'0 and water holdingcapacity (w.h.c.) 55 %, (b) clay (Rothamsted), pH7'2, w.h.c. 40 %, (c) sandy loam (Woburn, Beds.),pH 5'6, w.h.c, 47 % and (d) black fen peat (Felt-well, Norfolk), pH 5'8, w.h.c. 210 %. All soilswere air dried and sieved (4 mm) before use.

Except in Experiment 4, the same fungal isolate(30) was used throughout. It was grown on auto-claved wheat seeds at c. 20 DC and producedabundant pycnidia within about 3 weeks. Theseeds were soaked in water to produce a pycnidio-

0007-1536/79/2828-5300 $01.00 © 1979 The British Mycological Society

92 Survival of Phoma in soil

spore suspension which was calibrated using ahaemacytometer and then used to inoculate a knownweight of soil. After thorough mixing, the moisturecontent was adjusted to the required level and thesoils distributed to polythene bags (500 gauge)which were sealed and incubated in the dark atconstant temperature. When necessary, the mois-ture content was maintained at its original valueby the addition of sterile distilled water.

Sampling and testing of soils

On each sampling occasion the soil was well mixedand samples of c. 20 g withdrawn for testing usinga sterilized spatula. On most occasions, furthersamples were taken into glass tubes, weighed,dried at 105° for 24 h and then reweighed todetermine soil moisture content.

Tuber slice test. Soil samples were each spreadto five wounded slices of cv. Arran Banner pre-pared on the previous day (Hide, Griffith &Adams,1977). The percentage wounds rotting with gan-grene was recorded after incubation at 5° for8 weeks.

Soil dilution plates. Weighed samples (usually10 g) of soil were shaken for 2 h in 100 ml glass-distilled water, then diluted as appropriate and0'5 ml aliquots spread over four or five replicateagar plates. The two agar recipes usually usedwere modifications of that described by Mooi &Tichelaar (1965): Medium A was potato dextroseagar (Oxoid) containing 6 mg a.i./l pentachloro-nitrobenzene (PCNB), 20 mg a.i./l thiophanatemethyl and 10 mg/I Aureomycin (all added aspowders after autoclaving and cooling to c. 50°);Medium B was 10 gil malt extract (Oxoid) and12 gil agar (Oxoid NO.1) containing 2 mg a.i./lPCNB, 5 rng a.i./l thiophanate methyl and10 mg/I Aureomycin (PCNB and thiophanatemethyl added in a small volume of acetone,Aureomycin as powder after autoclaving and cool-ing). In Experiment 3, the recipe of McCracken& Logan (1977), but with Aretan at 7'5 mg pro-duct/I (0'225 mg a.i./l z-rnethoxyethylmercurychloride) was also used (Medium C). Colonies ofP. exigua var. fooeata were recognized by theproduction of anthraquinone pigment and crystals.The number of colonies developing on each platewas recorded after 7-10 days incubation at 20°.

RESULTS

Experiment 1

Autoclaved and non-autoclaved loam was inocu-lated on 10 April 1975, giving concentrations of1'01 x 106 (autoclaved) and 1'17 x 106 (non-auto-claved) spores/g dry weight of soil. Each soil was

divided into two equal parts, the moisture contentsof which were adjusted to 25 and 36 % dry weightand then the soils were subdivided and distributedto 5, 10 and 15°. Samples were taken at intervalsover 3 years for testing on slices and on dilutionplates (Medium A).

For statistical analysis, slice infection data weretransformed to logits and regressed on log time.There were highly significant effects of tempera-ture, autoclave and soil moisture treatments(P < 0'001) on the rate that populations declined.Persistence was longer in the drier soils, but theeffect of moisture was not large (Table 1). Foreach combination of autoclave and temperaturetreatments, the two moisture treatments of thenon-transformed data have therefore been averaged(Fig. 1A) and these show that in non-autoclavedsoils, populations reached undetectable levelswithin about 8 months at 15°, 2 years at 10° and3 years at 5°. In autoclaved soils populationspersisted longer but also declined, particularly at15°. Dilution plate counts were transformed tologarithms and an analysis of variance againshowed highly significant effects of temperatureand autoclave treatments (P < 0'001) but not ofsoil moisture. The results (Fig. 1B) were similarto the slice infection data but the method was lesssensitive, having a threshold of about 10z-1ea

propagules/g dry weight of soil below which thepathogen could not be reliably detected. In auto-claved soils, populations remained approximatelyconstant, or even increased slightly for 3-4 months(presumably because of colonization of sterileorganic matter) but later decreased at all tempera-tures as in non-autoclaved soils.

Experiment 2

Non-autoclaved loam was inoculated on 10 Feb.1976, giving a concentration of 2'1 x 105 spores/gdry weight of soil and then divided into three equalparts, the moisture contents of which were adjustedto 15, 29 and 42 % dry weight. The soils werefurther subdivided for incubation at 5, 10 and15° and samples were taken at intervals over 2years for testing on slices and on dilution plates(Medium B).

Both test methods showed the same trends andtherefore only slice infection data are presented.As in Experiment 1, logit transformations of infec-tion were regressed on log time. All regressionswere significant and analysis showed highly signifi-cant effects of temperature and soil moisture treat-ments (P < 0'001). From the regression lines, thetime taken for the population to fall to a levelcausing 50 % infection of slice wounds was esti-mated. The results (Table 1) confirm that survival

M.J. Adams 93

A

\00

" 75t;2.-~ 500::o

. ~;r.

~5~

50~O105~

L--,------r-=----.---.:~'"S__l_.:l~...:=:::%:~"'D_lJr100 150

B

:=::+

-- 3·0-::0

,:ijg. ~

fo'il 2·0::C~

ell

.§.~

2o:>

\ ·00

"~ I'".~ S.LD. (7 DY .)i3'0u

Time after inoculat ion (weeks )

Fig. 1. Decline in populations of Phoma exigua var. [oueata after inoculation into autoclaved or non-autoclaved loam (Experiment 1) as detected by infection of wounded potato slices (A) and by dilutionplates (B). Means of two moisture treatments, autoclaved loam incubated at 5 (0), 10 (ll.) and 15° (D),non-autoclaved loam at 5 (e), 10 (A) and 15° (.). Shaded area represents infection on uninoculatedcontrol slices.

94 Survival of Phoma in soil

15

23 (21-25)19 (17-21)

31 (29-34)18 (17-20)

3 (2-5)

10

42 (38-46)36 (32-4°)

65 (58-73)36 (33-39)13 (11-15)

5112 (94-138)*82 (69-101)

98 (88-115)65 (57-76)29 (26-33)

152942

Expt 1

Expt 2

Table 1. Time (weeks from inoculation) estimated for populations of P. exigua var. foveatain soil to decline to that causing 50 % infection of slice wounds

Soil Temperature (0C)moisture

(% dry wt)

2536

*Figures in parentheses are 95 % confidence limits.

A

B

60

Soil moisture ('1r water holding capacity)

80 100

Fig. 2. Populations of Phoma exigua var. foueata after inoculation into loam (0), clay (e), sandy loam(6) or peat (.) incubated at different moisture contents (Experiment 3). A, As detected 44 weeks afterinoculation by infection of wounded potato slices. (Bars are S.B.D.S for each soil type; 7 D.P. for loam,9 D.P. for other soils.) B, As detected 47 weeks after inoculation on dilution plates. (Bars are S.B.D.S foreach soil type; 28 D.P. for loam, 36 D.P. for other soils.)

M.J. Adams 95was better in the cooler soils and show a greaterpersistence in the drier soils at each temperature.There was a fairly good correspondence betweenthese figures and those for Experiment 1.

Experiment 3Non-autoclaved loam, clay, sandy-loam and peatwere inoculated on 25 March 1977 giving concen-trations respectively of'z-za x 105,2'22 x 105, 2'30 X

105 and 3'84 x 105 spores/g dry weight of soil.Samples of each soil were adjusted to nine moisturecontents varying from air dry to almost field cap-acity and all were incubated at 100. Two replicatebatches of five slices were inoculated after 36 and44 weeks, and samples were tested on dilutionplates after 45 weeks (Medium B) and 47 weeks(Medium C).

The different sampling methods and datesshowed a similar relationship between popula-tion and soil moisture. The slice infection resultsafter 44 weeks (Fig. 2A) show that survival wasbest in fairly dry soil (c. 25 % w.h.c.) and becamepoorer as moisture content was increased. Sur-vival was also poor in the driest soils used. Whenthe different soils were compared at similarmoisture contents (as % w.h.c.) there was noevidence that soil type had influenced longe-vity. When the results were compared withthose at 36 weeks it was evident that popula-tions were decreasing in all soils. The dilutionplate counts after 47 weeks (Fig. 2B) confirmedthe effect of soil moisture on survival, but, incontrast to the slide infection results, recoveryof the pathogen was lower from the loam andparticularly the sand than from the peat or claysoils. There was also an unexplained peak recovery

from the sand at c. 80 % w.h.c., which alsoappeared on the 45 week dilution plates but onneither of the slice samples.

Experiment 4

Non-autoclaved loam and clay were inoculatedon 25 March 1977with spores or mycelial maceratesof six isolates of P. exigua var. foveata made fromrots on tubers of different cultivars and sources.The spore concentration was 1'75 x 10'/g dryweight of soil and for the mycelial macerate thegrowth from two flasks of 100 ml z % malt extractliquid cultures, which had been incubated on anorbital shaker for 9 days at 200

, was bulked,blended into 50 ml distilled water and 20 mlliquidadded to 200 g soil. The moisture contents wereadjusted to 26 % (loam) or 23% (clay) dry weight(respectively 47 and 58 % w.h.c.) and all sampleswere incubated at 100. Two replicate batches offive slices were inoculated after 36 and 44 weeksand samples were tested on dilution plates(Medium B) after 36 weeks.

The results after 36 weeks (Table 2) showedthat populations were larger following mycelialthan spore inoculation. Populations were alsolarger (P < 0'001) in the loam than the clay,possibly because of direct or indirect effects ofdifferences in soil moisture content. Isolate 28,which had been maintained in culture for severalyears, had become weakly pathogenic and appearedto have much smaller populations than theother isolates. Other differences between isolates,although occasionally statistically significant didnot seem to be consistent. Slice infection at 44weeks showed that populations were decreasingbut were otherwise similar to those at 36 weeks.

Table 2. Soil populations ofP. exigua var. foveata assessed36 weeks after inoculation with spores or mycelialmacerates of six fungal isolates

Slice infection (angles)Dilution plate counts

(log, no colonies/mg dry wt)

Inoculum

Soil TypeIsolate

28*293°32

3334

S.H.D. (means)

Spores~

Loam Clay

72.6 49"653'8 39'262·6 29·647'2 36'561'3 30'3

±7'27

Mycelium~

Loam Clay

50'9 13'4go'o 84'2

9o'0 86'0go'o 80'2

9o'0 78'3go'o 80'2

±4'36

Spores~

Loam Clay

1'56 0'421'70 1'281'50 l'U

1'34 0'771'57 1'01

±0'139

Mycelium~

Loam Clay

0"91 0'4°1'94 0'952'02 1'162'14 1'131'46 0'881'88 0'90

±O'152

*Non sporing isolateproducing antibiotic 'E'; all others were E-ve (Logan & O'Neill, 1970).

REFERENCES

ANON. (1977). Potato gangrene (Phoma exigua var.foveata). Annual Report of Research and TechnicalWork of the Department of Agriculture, NorthernIrelandfor 1976, p. 151.

ATKINSON, R. G. (1954). Quantitative studies on thesurvival of fungi in five-year-old soil dried cultures.Canadian Journal of Botany 32, 673-678.

BAKERSPIGEL, A. (1953). Soil as a storage medium forfungi. Mycologia 45, 596-604.

BOEREMA, G. H. (1977). De veroorzakers van gangreenby aardappel. Gewasbescherming 8, 91-94.

BOEREMA, G. H. & HOWELER, L. H. (1967). Phomaexigua Desm. and its varieties. Persoonia 5, 15-28.

ENTWISTLE, A. R. (1971). The infection of potatoesbyPhoma exigua. Annals ofApplied Biology 69, 213-222.

Fox, R. A. & DASHWOOD, E. P. (1972). Potato gan-grene. Scottish Agriculture 51, 372-376.

Fox, R. A., DASHWOOD, E. P. & WILSON, H. M. (1976).Biology of potato gangrene. Annual Report of theScottish Horticultural Institute for 1975, pp, 48-5°.

Fox, R. A., DASHWOOD, E. P. & WILSON, H. M. (1977).Biology of potato gangrene. Annual Report of theScottish Horticultural Institute for 1976, pp. 54-56.

HIDE, G. A. (1978). Incidence of pathogenic fungi onScottish potato seed stocks derived from stem cut-tings. Potato Research 21, 277-289.

HIDE, G. A., GRIFFITH, R. L. & ADAMS, M. J. (1977).Methods of measuring the prevalence of Phomaexigua on potatoes and in soil. Annals of AppliedBiology 87, 7-15.

KHAN, A. A. & LOGAN, C. (1968). A preliminarystudyof the sources of potato gangrene infection. Euro-pean Potato Journalu, 77-87.

LOGAN, C. (1974). The effectof soil- and tuber-borneinoculum on the incidence of potato gangrene.Annals of Applied Biology 78, 251-259.

I thank Miss H. J. Davies and Miss M. A. Kempfor technical assistance and Mr R. Payne forstatistical advice.

Survival of Phoma in soil

P. exigua var. foveata without causing symptoms(Fox & Dashwood, 1972), but the importance ofthis for the long-term survival of the fungus isnot yet clear. Preliminary results at Rothamstedusing inoculated soil kept out-of-doors have sug-gested that the fungus survives for longer thanmight be expected from the results at constanttemperatures and moistures but the populationsnevertheless continue to decline.

It seems clear from these results and from fieldexperience, that with commercial crops, the seedof which frequently carries the pathogen, and withnormal rotations, the soil is unlikely to be a signifi-cant reservoir of the pathogen contributing toprogeny tuber contamination. However, in themultiplication of disease free seed stocks, even asmall population of the pathogen could be ofsignificance.

DISCUSSION

Temperature and moisture have been shown to beimportant factors affecting the survival of P. exiguavar. foueata in soil. The fungus survived best incool, dry soils and usually rather poorly when themoisture content exceeded about 50 % waterholding capacity, but survival was also poor in thedriest soils « 20 % w.h.c.), The form in whichthe fungus survives is uncertain. No sexual stageis known and the thin-walled pycnidiospores seemunlikely to survive for long periods. Probablythick-walled resting mycelium or chlamydospores,which can be seen in colonies on agar plates, areresponsible for the survival detected. Bacteriallysis at high soil moisture and desiccation in verydry conditions may be the factors that limitsurvival. The importance of other micro-organismsfor survival in the soil is illustrated by the resultsfrom autoclaved soil in Experiment 1, where thefungus survived much longer than in non-sterilizedsoil. Introduction of micro-organisms during thesampling may have contributed to the decline inpopulations in autoclaved soil, but it is noteworthythat Atkinson (1954) found that few Fungi Im-perfecti survived for as long as 5 years in air-drysterile soil in the laboratory. Also Bakerspigel(1953) reported that an unidentified Phoma sp.from Chrysanthemum survived for only about 4years in sterile loam and for shorter periods inother soil types.

The results illustrate the shortcomings of themethods available for assessing populations ofP. exigua var, fooeata in soil. The slice techniqueis sensitive but does not measure absolute con-centrations. Dilution plates give direct populationcounts but cannot detect small populations. Com-parison of plate and slice results in the same experi-ment sometimes reveals discrepancies, as inExperiment 3 where the plate counts suggested adifference between soil types which was not shownby the slices. Experience in other experimentsindicates that dilution plate counts are very sus-ceptible to other components of the soil microfloraand that the slice results would be more dependablefor comparisons between soil types.

The results reported in this paper can only beextrapolated to field soils with caution, sincesurvival may be influenced by fluctuations inmoisture and temperature, the presence of othercrop plants or weeds and the addition of freshorganic matter as plants die. Fluctuating conditionscould stimulate germination of resting structures,which might either decrease longevity if myceliumwere lysed or increase it if new resting structureswere formed. There are reports that the roots ofsome crop plants and weeds have been invaded by

M.J.Adams 97LOGAN, C. & O'NEILL, R. (1970). Production of an

antibiotic by Phoma exigua. Transactions of theBritish Mycological Society 55, 67-75.

McCRACKEN, A. R. & LOGAN, C. (1977). A selectivemedium for the isolation of Phoma exigua var.[ooeata from soil. Record of Agricultural Research,Ministry of Agriculture Northern Ireland 25, 71-76.

MALCOLMSON, J. F. & GRAY, E. G. (1968). Factorsaffecting the occurrence of gangrene (Phoma exigua)in potatoes. Annals of Applied Biology 62, 77-87.

MoOI, J. C. & TICHELAAR, G. M. (1975). Gangrene.Annual Report of the Institute of PhytopathologicalResearch, Wageningenfor 1974, pp. 29-30.

TODD, J. M. & ADAM, J. W. (1967). Potato gangrene:some interconnected sources and factors. Proceedingsof the Fourth British Insecticide and Fungicide Con-ference 1, 276-284.

(Accepted for publication 24 October 1978)

MYC 73