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The effects of temperature on the development of gangrene following laboratory inoculation of potato tubers with Phoma exigua var. foveata
Physiol.
Pl. Path.
(1971).
1, 477-487
The effects of temperature on the development of gangrene following laboratory inoculation of potato tubers with Phoma exigua var. foveata F’. A. LANGTON Plant Breeding (Acceptedfor
Institute, publication
Trumpington, May
Cambridge,
England
1971)
Potato tubers were inoculated with cores taken from agar cultures of Phoma exigua var. foueata. The relationship between the size of subsequently formed gangrene rots and time The relationship was influenced by of incubation typically showed a point of inflexion. incubation temperature possibly through two conflicting factors, fungal growth and tuber resistance. Both factors increase with temperature from 0 to 20 “C producing an acceleration of the early stages of rotting over the range 0 to 10 “C, an earlier limitation of rotting with rise in temperature, and increased variability at 15 and 20 “C. Some apparently conflicting results of temperature effects on rotting reported by other workers can be explained by this relationship. A seasonal effect was found in that the time of testing differentially affected rotting at 5 and 10 “C with a fixed incubation period of 28 days. Rotting was favoured by the lower temperature initially (July) and by the higher one later in the storage season (February). Tuber resistance declined with storage and the relationship between this and the differential rotting effect is discussed. The results are related to temperature relationships observed in agricultural practice and to tests of susceptibility to gangrene.
INTRODUCTION Potato gangrene is a storage disease which can cause severe losses. The causal fungus (Phoma exigua var. foveata) can infect tubers in the soil before harvest but the infections remain latent until later in the storage season when some develop into rots [IO]. Inoculum is also brought into the store on the tuber periderm and in adhering soil where, in both cases, it remains viable throughout storage [3]. In practice, gangrene is associated with tuber damage which probably introduces inoculum to exposed flesh tissue or pushes latent inoculum further into the flesh where it can initiate rotting [IO]. On the farm, low temperatures during storage are conducive to high levels of gangrene. This effect has been confirmed experimentally using naturally infected potato tubers [9]. It was also shown that damage caused by grading in cold conditions increased levels of disease. A further line of evidence has been put forward to substantiate this low temperature effect. Malcolmson [8] inoculated wounds made in the periderm of tubers with disks taken from plate cultures of the pathogen and stored the tubers at temperatures ranging from 5 to 28 “C for 42 days. Rots produced were very small but
478
F. A. Langton
were largest at the lowest temperature (5 “C) and decreased in size with rise of storage temperature. She found that all tubers showed rots at temperatures between 5 and 12 “C, some tubers between 14.5 and 20.3 “C but none at higher temperatures. Khan [Z] similarly inoculated tubers and stored these at temperatures ranging from 5 to 25 “C for 42 days and found the most extensive rots at 5 “C and progressively less extensive rots at higher temperatures, He found that all tubers showed rots at temperatures between 5 and 20 “C but that no rots were produced at 25 “C. Kranz [4] using an inoculation method where agar blocks from cultures of the pathogen were inserted into “V” wounds made in the periderm of tubers at temperatures from 4 to 26 “C found that temperature was inversely related to rot development in most tests with three out of four varieties after 42 days. With the fourth variety rots were generally most extensive at 10 “C. In the course of experiments to develop a laboratory test of tuber resistance to gangrene, and using a similar inoculation method, the present author obtained conflicting results [5]. In an experiment with eight potato varieties incubated at either 5 or 10 “C, there was rapid rotting at 10 “C and, averaging over varieties, a rot score of 13.6 mm after 13 days. At 5 “C, however, rots developed more slowly and it took 21 days for the rot score to reach 13.4 mm. The present paper gives the results of laboratory experiments made to investigate more fully the effects of temperature on rotting, in an attempt to resolve the differences reported above and to relate the results to temperature effects in agricultural practice. MATERIALS
AND
METHODS
All varieties of potatoes used in the experiments were grown under the same conditions at Cambridge and after harvest were stored under uniform conditions in an unheated but frost-free store. P. exigua var. foveata was cultured on malt extract agar with mycological peptone (Oxoid CM 59) at room temperature (18 to 23 “C). The isolate used was negative for substance “E” [7]. Washed tubers were inoculated by inserting cores (5 mm diameter) taken from leading edges of Petri dish cultures of the pathogen, in wounds (5 mm diameter) made through the periderm either with a limited depth (4.5 mm) cork-borer or twist drill. The former was used in the 1968-69 storage season and the latter subsequently. In both cases the “wells” were made by cutting and removing the unwanted tissue. Pieces of “Sellotape” were stuck over inoculation points for about 1 week after inoculation to prevent the inoculum from drying out quickly. Large tubers were used whenever possible so that more than one inoculation could be made on each to gain a better measure of reaction, since it had previously been shown that tuber size does not affect tuber reaction [5]. Positions well away from eyes were chosen to avoid inoculating into pith tissues. In the sampling experiments an uninoculated wound was included on each tuber to see if latent infections would be expressed. None were and in subsequent experiments uninoculated wounds were omitted. After inoculation tubers were placed in cooled incubators accurate to + 1 “C of the set temperature or in drawers in the laboratory at room temperature. The use of “Sellotape” ensured similar moisture conditions during initial infection but after removal no attempt was made to standardize humidities at the various temperatures used. The influence of this factor was investigated and is described later.
Temperature
effects
on gangrene
development
479
Measurements were made to give the average rot score per treatment (average lesion radius plus depth/2), which includes a constant (3.5 mm), the score for the original wound. In determining the mean score for a treatment any wounds apparently not infected were included in the calculations, thus combining the two parameters, number of successful inoculations and mean rot dimensions, used by both Malcolmson [S] and Khan [Z]. The scores for “unsuccessful” inoculations were, therefore, considered extreme values of the variable, rot size. Kranz [3] has described “Subinfektionen” where there is penetration of tissue by the fungus to a depth of one cell, and where there has not been failure to infect but rather limitation of fungal growth following infection. Such infections could not be seen without the aid of a microscope and would probably be classified by Malcolmson and Khan as unsuccessful inoculations. RESULTS
Sampling
experiments
During 1968-69 experiments were carried out in which tubers were sampled at various times after inoculation to follow the development of rots at different temperatures. In November, the varieties King Edward VII and Epicure were used and in the following March, King Edward VII and Majestic. On both occasions tubers were inoculated, each in two positions, and incubated at 0, 5, 10 and 15 “C and at room temperature. On each of four dates after inoculation five tubers per variety were taken at random from each of the differently incubated stocks and the gangrene score calculated. Rot development after inoculation is shown in Fig. 1. The graphs can be interpreted as portions of an inflected curve. At 5 “C in all four cases there was evidence of a lag phase in rot development followed by a period of more rapid development which then gradually slowed down. At 0 “C the lag phase was much more prolonged but only in the case of King Edward VII in the March experiment was rot development not progressive with time at the end of the experiment. At 10 “C far more rotting occurred in the period between inoculation and first sampling than at 5 “C and this was associated with an earlier onset of retardation. Thus at the first examination rots were always larger at 10 “C than at 5 “C. This was so for the second and third samplings also in the November experiment but in King Edward tubers final rots were larger at 5 “CL In the March experiment rots were larger at 5 “C at the second and subsequent samplings. Whether rots are larger at 5 or 10 “C may depend, therefore, on time of sampling, At room temperature there was little or no further rotting on Majestic tubers after the eighth day. This was so for some tubers of other varieties at room temperature but, in contrast, a number of lesions developed to a very large size. Development was, therefore, characterized by great variability. This is indicated in Fig. I where the ranges of tuber scores are shown. The development at 15 “C generally lay midway between that at 10 “C and that at room temperature, and again showed a tendency towards greater variability than at lower temperatures. Although rot extension in depth proceeded more rapidly than surface spread, there was no evidence of a differential effect of temperature.
480
F. A.
Langton
Sections through rots showed differences in appearance at the various temperatures. In all varieties lesions appeared moist at 0 and 5 “C at each sampling date, whilst at 15 “C and room temperature rots appeared dry and corky and were difficult to cut. The small rots which appeared to have stopped enlarging could usually be separated easily with the finger nail from neighbouring and apparently healthy tissue. This was never so for rots formed at low temperatures. In general, at 10 “C, lesions appeared moist at the first sampling but some corkiness later developed. At all temperatures, rotted tissue tended to extend initially in a geometrically regular manner into the tuber but later, and particularly at high temperatures, rots assumed
Room temperature
t
IOOC
5oc
WC t
t
15OC t
(18 to 23OC)
t King Edward VII (November 1
Epicure (November)
King Edward VII (March)
Majestic (March)
Tie
FIG. 1. The development indicate the ranges of tuber inoculation wound.
after
inoculation (days1 _____f
of gangrene rots scores; the horizontal
at a range of temperatures. Vertical bars dotted lines indicate the score of the initial
Temperature
effects
on gangrene
481
development
very irregular shapes often following the pith tissue. Cavities were formed at all temperatures and towards the end of the experiments at 10 “C and above pycnidia of P. exigua var. foveata could be seen lining these, as well as pushing through rotted periderm tissue.
In 1969-70 varietal experiments were carried out in July, September and February, to see if the time of year at which testing is carried out has an effect on rotting at 5 and 10 “C. On each occasion 20 tubers per variety were inoculated, each in three positions, and incubated for 28 days. Half the tubers were maintained at 5 “C and half at 10 “C. In the July experiment five first and second early varieties were compared but in the two later ones these were supplemented with a further early and six maincrop varieties. The results, averaged over varieties, are shown in Table 1. Details of varietal reaction at 10 “C have been given elsewhere [5]. TABLE
Gangrene
sores
after incubation for
1
28 days at 5 and 10 “C on three separate 1969-70
occasions
during
Difference
Time
of
testing
July September February
Mean
score
in rot score showing differences at P = 0.05) “C 5 < 10 “C Not different
(percentage varieties
(mm)
(averaging over varieties) 5 “C
10 “C
IO.82 14.79 14.55
a.77 14.76 16.66
5>10 80 17 0
0 25 100
20 58 0
The time of testing affected rotting at the two temperatures differentially. Early in the storage season rotting was greater at 5 “C, with a fixed incubation time of 28 days, whilst later the situation was reversed. Rots tended to be rather corky at 10 “C but not at 5 “C, particularly in July. Rots produced at both 5 and 10 “C in September were larger than those produced in July. The comparison, using only the early maturing varieties, gives a more pronounced difference with respective September figures at 5 and 10 “C of 15.55 and 16.06 mm. This trend of lessening resistance continued to February at 10 “C but not at 5 “C. The relationship between this effect and the differential temperature effect is discussed below. Varietal
inJuences
To determine how variety influences the relationship between lesion size and time of incubation, a sampling experiment was carried out in January/February 1969 with five varieties considered to differ in susceptibility in agricultural practice. The varieties used were two resistant maincrops, Arran Consul and King Edward VII, two susceptible maincrops, Majestic and Pentland Falcon, and a susceptible early, Ulster Chieftain. Tubers were each inoculated in two positions and incubated at either 5 or 10 “C. Five-tuber samples were scored on four occasions after inoculation. The development of rots at the two temperatures ii shown in Fig. 2.
482
F. A. Langton
At each sampling date the mean gangrene score averaged over varieties was larger at 10 “C and at the end of the experiment, when rot scores for at least Ulster Chieftain at 10 “C were being limited by tuber size, there was apparently no trend towards larger rots at 5 “C. The difference between mean scores for the two temperatures at the final samplings was 3.70 mm whilst at the first it was 3.36 mm showing that the major difference lay in the development between inoculation and first sampling. 30 5 T
Time after
FIG. 2. The development Vertical bars indicate LAD. O-O, King Edward l - . -. -0, Ulster Chieftain.
inoculation (days) ---+-
of gangrene rots on tubers of five varieties at 5 and 10 “C. l - -0, Arran Consul; values for varietal means at P = 0.05. VII; O-----O, Majestic; l . . . . . ..O. Pentland Falcon;
There was similar varietal ranking at the two temperatures, with Arran Consul bearing the smallest rots at all examinations and Ulster Chieftain the largest. A was consistent significant difference (P < O-05) in rot size for these two varieties shown at 5 and 10 “C except at the first examination at 5 “C when there was a distinct lag phase affecting all varieties. The period between first and second sampling at 5 “C, when relatively rapid rotting occurred, revealed a clear difference between Ulster Chieftain and Arran Consul and subsequently, as rot development in all varieties slowed down, the difference became accentuated. As at 5 “C, tuber rots on all the varieties at 10 “C showed similar patterns of growth, and lag phases were not detected. Rots on Arran Consul appeared to show an earlier onset of retardation than those on Ulster Chieftain, and this varietal difference increased with time. The difference at the final examination is probably spuriously small since rots on Ulster Chieftain were limited by tuber size. At 5 and 10 “C, however, standard error also increased, approximately in proportion to the varietal difference. These results are later discussed in relation to the assessment of gangrene susceptibility.
Temperature
effects
Temperature-growth
on gangrene
483
development
rate relationship
for P. exigua
var. foveata
To help in the interpretation of some of the results, growth rates of the culture of P. exigua var. foveata used were determined at different temperatures on malt extract agar with mycological peptone (Oxoid CM 59). Petri dishes were inoculated with cores taken with a 5 mm diameter cork borer from growing edges of cultures of the fungus and incubated at 0, 5, 10, 15, 20 and 25 “C, there being two dishes per temperature. After an initial lag phase fungal growth was linear. At 24 h intervals measurements were made along pre-marked diameters and for each temperature the growth rate was calculated as the regression coefficient of culture radius on time for the period of linear growth. The coefficient is a direct measure of growth rate using all the data. The results in Table 2 show that the fastest growth rate was recorded at 20 “C. The linear growth was in contrast to the pattern of size increase of tuber rots. TABLE
Radial
growth
of Phoma
exigua
Temperature
(“C)
Growth
0 5 10 15 20 25 Growth
rates
Effect of humidity
calculated
as regression
2
var. foveata
in culture rate
at da&ent
temperatures
(mm/hr)
0.03 0.08 0.14 0.17 0.22 0.05 coefficients.
on rot development
Ilumidity was not standardized in the incubators used in these studies. Several workers have reported, however, that humidity has an effect on rot development following inoculation. Malcolmson [8], for example, found that rots were larger when tubers were kept relatively dry, whilst Khan [.?I found that high humidity encouraged rot development in the cortex and that low humidity induced deeper penetration into the pith. Accordingly an experiment was carried out to determine whether humidity had any effect on rot size following the “Sellotape” inoculation method. Tubers of King Edward VII and Ulster Sceptre were inoculated in three positions using the “Sellotape” method, and 20 of each variety were placed in incubators at 4, 10 and 20 “C. In each incubator half the tubers were stored in dry open seedtrays whilst the remainder were stored in seed-trays with a layer of moist vermiculite and covered by propagator hoods which maintained high humidity conditions. Differences found between samples in the high humidity trays reflected temperature differences unaffected by humidity differences. Mean relative humidity values in the incubators were 72, 75 and 35% respectively for the temperatures 4, 10 and 20 OC, but the relative humidity in the covered seed trays was approximately 100%. There was no significant (P
484
F. A. Langton
Table 3 also shows, averaging over humidities, that tubers of the very susceptible Ulster Sceptre showed significantly (P < O*OOl) larger rots than those of King Edward at 4 and 10 “C but smaller ones (PC 0.001) at 20 “C. Rots produced on tubers of Ulster Sceptre at 10 “C were significantly (PC O-001) larger than those at 4 “C and those at 20 “C were significantly (P < O-001) smaller than those produced at either of the two lower temperatures. King Edward tubers, on the other hand, showed significantly (PC 0.01) larger rots at 20 “C with no difference between those formed at5andlO”C. TABLE
The effect of relative
Variety Ulster
Kinz
Temperature
Sceptre
Edward
VII
L.S.D.
5%
(“C)
4 10 20 4 10 20 between
3
on rot deuelopment for King temperatures
humidity
humidities
High
relative
Edward
Tuber humidity
VII and Ulster
scores (mm) Lower relative
17.10 19.92 12.45 14.07 14.41 16.83 within
temperatures:
Sceptre at di$erent
humidity
16.36 20.31 13.62 14.29 14.68 16.08 Ulster
Sceptre,
2.30;
King
Mean 16.73 20.12 13.03 14.18 14.55 16.46
Edward,
1.98.
DISCUSSION The gangrene score is designed to measure the average distance that the fungus grows into the potato tissue from the inoculation point and, since fungal growth in culture is linear, it might be expected to increase linearly with time. However, the relationship between rot size and length of incubation in the sampling experiments typically appeared inflected. The lag phase, which was most pronounced at the lowest temperatures, presumably covered the period prior to and during infection and the time during which early growth of the fungus could not be detected. The phase of retardation indicated that a factor was limiting fungal growth. This probably was not due to exhaustion of substrates since it became apparent at different stages of rotting, depending on the temperature. However, the possibility that temperature influences the availability of substrates cannot be ruled out. In general as the temperature was increased the limitation became more apparent. On the other hand, some very large rots were produced at room temperature. The temperature effects may be most simply explained by the interaction of two conflicting factors. The first is fungal growth which was found to be fastest at 20 “C in culture, an optimum similar to those found by other workers, for example Kranz [3]. The second is tuber resistance which we can postulate to increase with rising temperature to, at least, 20 “C. These factors together could produce the observed effects of acceleration of the early stages of rotting and earlier limitation when the temperature is raised from 0 to 10 “C. Tuber resistance would cause the cessation of rotting in the variety Majestic after 8 days at room temperature, and the interaction of the two factors would give rise to the extreme variability shown by the other varieties at this temperature. It is tempting to suppose that the corky appearance
Temperature
effects
on gangrene
development
485
which rots generally show at the higher temperatures is evidence of a resistance mechanism but there is no direct evidence to support this. It was generally found, as in earlier studies [5], that rots extend radially through the cortical tissues less than they do in depth into the medulla tissues. For example in the humidity experiment averaging over humidities, temperatures and varieties, the mean radial extension was 1 l-61 mm whilst the mean depth was 20.07 mm. It was also observed that rots extend symmetrically only at low temperatures and tend to follow the path of pith tissue at higher. This observation is evidence for differential tissue resistance and since it is more pronounced at higher temperatures is most likely not brought about by differences in nutrient status. As noted earlier, however, the possibility that temperature influences the availability of substrate cannot be ignored. Kranz [3] also concluded that the inner tissues were more susceptible to fungal growth. The mechanism of wound healing has been proposed to limit gangrene disease in potatoes (for example references [S, S]), and the temperature relations of this process [I] are similar to those proposed for the resistance mechanism in this paper. However, Kranz [3] has demonstrated that although wound healing will render wounded surfaces resistant to infection by the pathogen, it is unlikely to be effective in limiting fungal growth through the tuber tissues. He suggested that fungal growth is limited by another, as yet unknown, mechanism which is localized in the cortical tissues. It is thus possible that both mechanisms are operative on the farm and, if so, extreme care should be exercised in relating the laboratory findings to farming practice since in the laboratory the use of agar disks with growing mycelium and covering all inoculated wounds with “Sellotape” imposes conditions such that the pathogen always effects initial penetration and the observed resistance is that which limits fungal extension. Laboratory inoculation does, however, give reactions which correlate with susceptibilities found in agricultural practice [5]. Temperature and humidity will, no doubt, influence the process of infection but “Sellotape” ensures high humidity conditions in all cases. The acceleration of the early stages of rotting with rising temperature over the range 0 to 10 “C shown in all experiments would explain larger rots being found on tubers at 10 “C than at 5 “C when scored soon after inoculation. The tendency for limitation to be shown soonest at the higher temperature, however, means that with increasing length of incubation there would be an increasing possibility that rots would be larger at 5 “C than at 10 “C. This effect may explain the conflicting temperature relations reported in the literature since Langton [5] scored rots 13 and 21 days after inoculation and found growth more rapid at 10 “C, whilst Malcolmson [8] and Khan [Z] scored much later, after 42 days, and found the largest rots at 5 “C. Kranz [4], however, who also scored after 42 days, frequently found the largest rots at 10 “C. It has generally been reported that rotting decreases with increasing temperature over the range 5 to 20 “C, when scoring is after 42 days [Z, 4, 81. This relationship was shown by Majestic in the March sampling experiment in the present study but not by the other varieties. It is probable that many other factors, including time of testing, will modify the temperature effects. Thus, for example, the comparison between the results of the November and March sampling experiments will be influenced by the storage
486
F. A. Langton
conditions of the tubers before the samplings. The seasonal influence experiments showed that at both 5 and 10 “C rots became larger as the season advanced which is evidence of a progressive decline of tuber resistance (see also references [3, 8, lo]). The fall was particularly marked when testing was at 10 “C and at both temperatures was most pronounced between July and September. The fall is related possibly to the differential effects found; in July, when there was probably a high level of resistance, rots were largest at 5 “C, at which temperature resistance is poorly expressed, but later (February) when there might have been less resistance, rots were largest at 10 “C, the temperature favouring fungal growth. However, King Edward tubers in the sampling experiments gave results which cannot be explained on the basis of this relationship since, at 10 “C in March, smaller rots were produced after 28 days than in the previous November. This discrepancy remains unexplained. The five variety experiment gave disease susceptibility ratings in fair agreement with farm experience at both 5 and 10 “C with Arran Consul resistant, Ulster Chieftain susceptible, and the remaining three varieties reacting similarly in an intermediate position. Earlier work [5] has shown, however, that for acceptable varietal differentiation greater replication is required and better agreement might have been achieved in the experiment reported here had more tubers been used. Extensive use of this test method has shown, however, that King Edward VII does not react consistently as more resistant than Majestic although usually it appears so. Arran Consul does however, consistently appear resistant and Ulster Chieftain susceptible. It is, however, to be expected that varietal susceptibility in agricultural practice will be influenced by factors other than fungal extension through tuber tissues. Such factors may include susceptibility of tubers to damage and the ease with which the fungus can infect tubers. Varietal differences agreeing with those found in agricultural practice are shown more accurately in tests at 10 “C rather than at 5 “C [S]. The humidity experiment showed that high temperatures (20 “C) can give results in very poor agreement with farm experience. In the variety experiment variation increased with rot size to such an extent that although varietal means became more widely separated, varietal differences were no more sensitively detected with time once the 7 day stage had been passed at 10 “C and the 18 day stage at 5 “C. Incubation periods of about 28 days are used in varietal tests, but it appears that this could be reduced with no loss of effectiveness.
REFERENCES 1. ARTSCHWAGER,
E. (1927).
Wound
periderm formation
in the potato as affected by temperature.
3. ugric. Res. 35, 995-1000.
2. KHAN,
A. A. (1967). A study of some factors affecting infection and development of potato gangrene. Rec. agric. Res. Minist. Agtic. Nth. Ire. 16, 97-101. 3. KRANZ, J. (1958). Untersuchungen tiber die Phoma-Ftiule der Kartoffelknolle unter besonderer Berticksichtigung des Wirt-Parasit-Verhaltnisses. Phytopath. <. 33, 153-196. 4. KRANZ, J. (1959). tfber die Ausbreitung der Phoma-F&de der Kartoffelknolle in Abhingigkeit von Umweltfaktoren. Phytopath. <. 36, 101-l 10. 5. LANGTON, F. A. (1971). The development of a laboratory test for assessing potato varietal susceptibility to gangrene caused by Phoma exigua var. foveata. Potato Res. 14,29-38. 6. LOGAN, C. (1967). Potato gangrene. 3ournal of the National Association of Seed Potato Merchants
7, 78-80.
Temperature
effects
on gangrene
development
487
7. LOGAN, C. & KHAN, A. A. (1969). Comparative studies of Phoma spp. associated with potato gangrene. in Northern Ireland. Trans. Br. mycol. Sot. 52, 9-17. 8. MALCOLMSON, .I. F. (1958). Some factors affecting the occurrence and development in potatoes of gangrene caused by’ Phoma solanicola Prill. &-Delacr. Ann. a&l. Biol. 46, 639-650. 9. MALCOLMSON. 1. F. & GRAY, E. G. (1968). The incidence of eanerene of notatoes caused bv Phoma e.@$ in relation to handling and storage. Ann. appl. I&1.-62, 89-161. 10. TODD, J. M. & ADAM, J. W. (1967). Potato gangrene: some interconnected sources and factors. Proceedings of the 4th British Insecticide and Fungicide Conference 1967, pp. 276-284.
Pl. Path.
(1971).
1, 477-487
The effects of temperature on the development of gangrene following laboratory inoculation of potato tubers with Phoma exigua var. foveata F’. A. LANGTON Plant Breeding (Acceptedfor
Institute, publication
Trumpington, May
Cambridge,
England
1971)
Potato tubers were inoculated with cores taken from agar cultures of Phoma exigua var. foueata. The relationship between the size of subsequently formed gangrene rots and time The relationship was influenced by of incubation typically showed a point of inflexion. incubation temperature possibly through two conflicting factors, fungal growth and tuber resistance. Both factors increase with temperature from 0 to 20 “C producing an acceleration of the early stages of rotting over the range 0 to 10 “C, an earlier limitation of rotting with rise in temperature, and increased variability at 15 and 20 “C. Some apparently conflicting results of temperature effects on rotting reported by other workers can be explained by this relationship. A seasonal effect was found in that the time of testing differentially affected rotting at 5 and 10 “C with a fixed incubation period of 28 days. Rotting was favoured by the lower temperature initially (July) and by the higher one later in the storage season (February). Tuber resistance declined with storage and the relationship between this and the differential rotting effect is discussed. The results are related to temperature relationships observed in agricultural practice and to tests of susceptibility to gangrene.
INTRODUCTION Potato gangrene is a storage disease which can cause severe losses. The causal fungus (Phoma exigua var. foveata) can infect tubers in the soil before harvest but the infections remain latent until later in the storage season when some develop into rots [IO]. Inoculum is also brought into the store on the tuber periderm and in adhering soil where, in both cases, it remains viable throughout storage [3]. In practice, gangrene is associated with tuber damage which probably introduces inoculum to exposed flesh tissue or pushes latent inoculum further into the flesh where it can initiate rotting [IO]. On the farm, low temperatures during storage are conducive to high levels of gangrene. This effect has been confirmed experimentally using naturally infected potato tubers [9]. It was also shown that damage caused by grading in cold conditions increased levels of disease. A further line of evidence has been put forward to substantiate this low temperature effect. Malcolmson [8] inoculated wounds made in the periderm of tubers with disks taken from plate cultures of the pathogen and stored the tubers at temperatures ranging from 5 to 28 “C for 42 days. Rots produced were very small but
478
F. A. Langton
were largest at the lowest temperature (5 “C) and decreased in size with rise of storage temperature. She found that all tubers showed rots at temperatures between 5 and 12 “C, some tubers between 14.5 and 20.3 “C but none at higher temperatures. Khan [Z] similarly inoculated tubers and stored these at temperatures ranging from 5 to 25 “C for 42 days and found the most extensive rots at 5 “C and progressively less extensive rots at higher temperatures, He found that all tubers showed rots at temperatures between 5 and 20 “C but that no rots were produced at 25 “C. Kranz [4] using an inoculation method where agar blocks from cultures of the pathogen were inserted into “V” wounds made in the periderm of tubers at temperatures from 4 to 26 “C found that temperature was inversely related to rot development in most tests with three out of four varieties after 42 days. With the fourth variety rots were generally most extensive at 10 “C. In the course of experiments to develop a laboratory test of tuber resistance to gangrene, and using a similar inoculation method, the present author obtained conflicting results [5]. In an experiment with eight potato varieties incubated at either 5 or 10 “C, there was rapid rotting at 10 “C and, averaging over varieties, a rot score of 13.6 mm after 13 days. At 5 “C, however, rots developed more slowly and it took 21 days for the rot score to reach 13.4 mm. The present paper gives the results of laboratory experiments made to investigate more fully the effects of temperature on rotting, in an attempt to resolve the differences reported above and to relate the results to temperature effects in agricultural practice. MATERIALS
AND
METHODS
All varieties of potatoes used in the experiments were grown under the same conditions at Cambridge and after harvest were stored under uniform conditions in an unheated but frost-free store. P. exigua var. foveata was cultured on malt extract agar with mycological peptone (Oxoid CM 59) at room temperature (18 to 23 “C). The isolate used was negative for substance “E” [7]. Washed tubers were inoculated by inserting cores (5 mm diameter) taken from leading edges of Petri dish cultures of the pathogen, in wounds (5 mm diameter) made through the periderm either with a limited depth (4.5 mm) cork-borer or twist drill. The former was used in the 1968-69 storage season and the latter subsequently. In both cases the “wells” were made by cutting and removing the unwanted tissue. Pieces of “Sellotape” were stuck over inoculation points for about 1 week after inoculation to prevent the inoculum from drying out quickly. Large tubers were used whenever possible so that more than one inoculation could be made on each to gain a better measure of reaction, since it had previously been shown that tuber size does not affect tuber reaction [5]. Positions well away from eyes were chosen to avoid inoculating into pith tissues. In the sampling experiments an uninoculated wound was included on each tuber to see if latent infections would be expressed. None were and in subsequent experiments uninoculated wounds were omitted. After inoculation tubers were placed in cooled incubators accurate to + 1 “C of the set temperature or in drawers in the laboratory at room temperature. The use of “Sellotape” ensured similar moisture conditions during initial infection but after removal no attempt was made to standardize humidities at the various temperatures used. The influence of this factor was investigated and is described later.
Temperature
effects
on gangrene
development
479
Measurements were made to give the average rot score per treatment (average lesion radius plus depth/2), which includes a constant (3.5 mm), the score for the original wound. In determining the mean score for a treatment any wounds apparently not infected were included in the calculations, thus combining the two parameters, number of successful inoculations and mean rot dimensions, used by both Malcolmson [S] and Khan [Z]. The scores for “unsuccessful” inoculations were, therefore, considered extreme values of the variable, rot size. Kranz [3] has described “Subinfektionen” where there is penetration of tissue by the fungus to a depth of one cell, and where there has not been failure to infect but rather limitation of fungal growth following infection. Such infections could not be seen without the aid of a microscope and would probably be classified by Malcolmson and Khan as unsuccessful inoculations. RESULTS
Sampling
experiments
During 1968-69 experiments were carried out in which tubers were sampled at various times after inoculation to follow the development of rots at different temperatures. In November, the varieties King Edward VII and Epicure were used and in the following March, King Edward VII and Majestic. On both occasions tubers were inoculated, each in two positions, and incubated at 0, 5, 10 and 15 “C and at room temperature. On each of four dates after inoculation five tubers per variety were taken at random from each of the differently incubated stocks and the gangrene score calculated. Rot development after inoculation is shown in Fig. 1. The graphs can be interpreted as portions of an inflected curve. At 5 “C in all four cases there was evidence of a lag phase in rot development followed by a period of more rapid development which then gradually slowed down. At 0 “C the lag phase was much more prolonged but only in the case of King Edward VII in the March experiment was rot development not progressive with time at the end of the experiment. At 10 “C far more rotting occurred in the period between inoculation and first sampling than at 5 “C and this was associated with an earlier onset of retardation. Thus at the first examination rots were always larger at 10 “C than at 5 “C. This was so for the second and third samplings also in the November experiment but in King Edward tubers final rots were larger at 5 “CL In the March experiment rots were larger at 5 “C at the second and subsequent samplings. Whether rots are larger at 5 or 10 “C may depend, therefore, on time of sampling, At room temperature there was little or no further rotting on Majestic tubers after the eighth day. This was so for some tubers of other varieties at room temperature but, in contrast, a number of lesions developed to a very large size. Development was, therefore, characterized by great variability. This is indicated in Fig. I where the ranges of tuber scores are shown. The development at 15 “C generally lay midway between that at 10 “C and that at room temperature, and again showed a tendency towards greater variability than at lower temperatures. Although rot extension in depth proceeded more rapidly than surface spread, there was no evidence of a differential effect of temperature.
480
F. A.
Langton
Sections through rots showed differences in appearance at the various temperatures. In all varieties lesions appeared moist at 0 and 5 “C at each sampling date, whilst at 15 “C and room temperature rots appeared dry and corky and were difficult to cut. The small rots which appeared to have stopped enlarging could usually be separated easily with the finger nail from neighbouring and apparently healthy tissue. This was never so for rots formed at low temperatures. In general, at 10 “C, lesions appeared moist at the first sampling but some corkiness later developed. At all temperatures, rotted tissue tended to extend initially in a geometrically regular manner into the tuber but later, and particularly at high temperatures, rots assumed
Room temperature
t
IOOC
5oc
WC t
t
15OC t
(18 to 23OC)
t King Edward VII (November 1
Epicure (November)
King Edward VII (March)
Majestic (March)
Tie
FIG. 1. The development indicate the ranges of tuber inoculation wound.
after
inoculation (days1 _____f
of gangrene rots scores; the horizontal
at a range of temperatures. Vertical bars dotted lines indicate the score of the initial
Temperature
effects
on gangrene
481
development
very irregular shapes often following the pith tissue. Cavities were formed at all temperatures and towards the end of the experiments at 10 “C and above pycnidia of P. exigua var. foveata could be seen lining these, as well as pushing through rotted periderm tissue.
In 1969-70 varietal experiments were carried out in July, September and February, to see if the time of year at which testing is carried out has an effect on rotting at 5 and 10 “C. On each occasion 20 tubers per variety were inoculated, each in three positions, and incubated for 28 days. Half the tubers were maintained at 5 “C and half at 10 “C. In the July experiment five first and second early varieties were compared but in the two later ones these were supplemented with a further early and six maincrop varieties. The results, averaged over varieties, are shown in Table 1. Details of varietal reaction at 10 “C have been given elsewhere [5]. TABLE
Gangrene
sores
after incubation for
1
28 days at 5 and 10 “C on three separate 1969-70
occasions
during
Difference
Time
of
testing
July September February
Mean
score
in rot score showing differences at P = 0.05) “C 5 < 10 “C Not different
(percentage varieties
(mm)
(averaging over varieties) 5 “C
10 “C
IO.82 14.79 14.55
a.77 14.76 16.66
5>10 80 17 0
0 25 100
20 58 0
The time of testing affected rotting at the two temperatures differentially. Early in the storage season rotting was greater at 5 “C, with a fixed incubation time of 28 days, whilst later the situation was reversed. Rots tended to be rather corky at 10 “C but not at 5 “C, particularly in July. Rots produced at both 5 and 10 “C in September were larger than those produced in July. The comparison, using only the early maturing varieties, gives a more pronounced difference with respective September figures at 5 and 10 “C of 15.55 and 16.06 mm. This trend of lessening resistance continued to February at 10 “C but not at 5 “C. The relationship between this effect and the differential temperature effect is discussed below. Varietal
inJuences
To determine how variety influences the relationship between lesion size and time of incubation, a sampling experiment was carried out in January/February 1969 with five varieties considered to differ in susceptibility in agricultural practice. The varieties used were two resistant maincrops, Arran Consul and King Edward VII, two susceptible maincrops, Majestic and Pentland Falcon, and a susceptible early, Ulster Chieftain. Tubers were each inoculated in two positions and incubated at either 5 or 10 “C. Five-tuber samples were scored on four occasions after inoculation. The development of rots at the two temperatures ii shown in Fig. 2.
482
F. A. Langton
At each sampling date the mean gangrene score averaged over varieties was larger at 10 “C and at the end of the experiment, when rot scores for at least Ulster Chieftain at 10 “C were being limited by tuber size, there was apparently no trend towards larger rots at 5 “C. The difference between mean scores for the two temperatures at the final samplings was 3.70 mm whilst at the first it was 3.36 mm showing that the major difference lay in the development between inoculation and first sampling. 30 5 T
Time after
FIG. 2. The development Vertical bars indicate LAD. O-O, King Edward l - . -. -0, Ulster Chieftain.
inoculation (days) ---+-
of gangrene rots on tubers of five varieties at 5 and 10 “C. l - -0, Arran Consul; values for varietal means at P = 0.05. VII; O-----O, Majestic; l . . . . . ..O. Pentland Falcon;
There was similar varietal ranking at the two temperatures, with Arran Consul bearing the smallest rots at all examinations and Ulster Chieftain the largest. A was consistent significant difference (P < O-05) in rot size for these two varieties shown at 5 and 10 “C except at the first examination at 5 “C when there was a distinct lag phase affecting all varieties. The period between first and second sampling at 5 “C, when relatively rapid rotting occurred, revealed a clear difference between Ulster Chieftain and Arran Consul and subsequently, as rot development in all varieties slowed down, the difference became accentuated. As at 5 “C, tuber rots on all the varieties at 10 “C showed similar patterns of growth, and lag phases were not detected. Rots on Arran Consul appeared to show an earlier onset of retardation than those on Ulster Chieftain, and this varietal difference increased with time. The difference at the final examination is probably spuriously small since rots on Ulster Chieftain were limited by tuber size. At 5 and 10 “C, however, standard error also increased, approximately in proportion to the varietal difference. These results are later discussed in relation to the assessment of gangrene susceptibility.
Temperature
effects
Temperature-growth
on gangrene
483
development
rate relationship
for P. exigua
var. foveata
To help in the interpretation of some of the results, growth rates of the culture of P. exigua var. foveata used were determined at different temperatures on malt extract agar with mycological peptone (Oxoid CM 59). Petri dishes were inoculated with cores taken with a 5 mm diameter cork borer from growing edges of cultures of the fungus and incubated at 0, 5, 10, 15, 20 and 25 “C, there being two dishes per temperature. After an initial lag phase fungal growth was linear. At 24 h intervals measurements were made along pre-marked diameters and for each temperature the growth rate was calculated as the regression coefficient of culture radius on time for the period of linear growth. The coefficient is a direct measure of growth rate using all the data. The results in Table 2 show that the fastest growth rate was recorded at 20 “C. The linear growth was in contrast to the pattern of size increase of tuber rots. TABLE
Radial
growth
of Phoma
exigua
Temperature
(“C)
Growth
0 5 10 15 20 25 Growth
rates
Effect of humidity
calculated
as regression
2
var. foveata
in culture rate
at da&ent
temperatures
(mm/hr)
0.03 0.08 0.14 0.17 0.22 0.05 coefficients.
on rot development
Ilumidity was not standardized in the incubators used in these studies. Several workers have reported, however, that humidity has an effect on rot development following inoculation. Malcolmson [8], for example, found that rots were larger when tubers were kept relatively dry, whilst Khan [.?I found that high humidity encouraged rot development in the cortex and that low humidity induced deeper penetration into the pith. Accordingly an experiment was carried out to determine whether humidity had any effect on rot size following the “Sellotape” inoculation method. Tubers of King Edward VII and Ulster Sceptre were inoculated in three positions using the “Sellotape” method, and 20 of each variety were placed in incubators at 4, 10 and 20 “C. In each incubator half the tubers were stored in dry open seedtrays whilst the remainder were stored in seed-trays with a layer of moist vermiculite and covered by propagator hoods which maintained high humidity conditions. Differences found between samples in the high humidity trays reflected temperature differences unaffected by humidity differences. Mean relative humidity values in the incubators were 72, 75 and 35% respectively for the temperatures 4, 10 and 20 OC, but the relative humidity in the covered seed trays was approximately 100%. There was no significant (P
484
F. A. Langton
Table 3 also shows, averaging over humidities, that tubers of the very susceptible Ulster Sceptre showed significantly (P < O*OOl) larger rots than those of King Edward at 4 and 10 “C but smaller ones (PC 0.001) at 20 “C. Rots produced on tubers of Ulster Sceptre at 10 “C were significantly (PC O-001) larger than those at 4 “C and those at 20 “C were significantly (P < O-001) smaller than those produced at either of the two lower temperatures. King Edward tubers, on the other hand, showed significantly (PC 0.01) larger rots at 20 “C with no difference between those formed at5andlO”C. TABLE
The effect of relative
Variety Ulster
Kinz
Temperature
Sceptre
Edward
VII
L.S.D.
5%
(“C)
4 10 20 4 10 20 between
3
on rot deuelopment for King temperatures
humidity
humidities
High
relative
Edward
Tuber humidity
VII and Ulster
scores (mm) Lower relative
17.10 19.92 12.45 14.07 14.41 16.83 within
temperatures:
Sceptre at di$erent
humidity
16.36 20.31 13.62 14.29 14.68 16.08 Ulster
Sceptre,
2.30;
King
Mean 16.73 20.12 13.03 14.18 14.55 16.46
Edward,
1.98.
DISCUSSION The gangrene score is designed to measure the average distance that the fungus grows into the potato tissue from the inoculation point and, since fungal growth in culture is linear, it might be expected to increase linearly with time. However, the relationship between rot size and length of incubation in the sampling experiments typically appeared inflected. The lag phase, which was most pronounced at the lowest temperatures, presumably covered the period prior to and during infection and the time during which early growth of the fungus could not be detected. The phase of retardation indicated that a factor was limiting fungal growth. This probably was not due to exhaustion of substrates since it became apparent at different stages of rotting, depending on the temperature. However, the possibility that temperature influences the availability of substrates cannot be ruled out. In general as the temperature was increased the limitation became more apparent. On the other hand, some very large rots were produced at room temperature. The temperature effects may be most simply explained by the interaction of two conflicting factors. The first is fungal growth which was found to be fastest at 20 “C in culture, an optimum similar to those found by other workers, for example Kranz [3]. The second is tuber resistance which we can postulate to increase with rising temperature to, at least, 20 “C. These factors together could produce the observed effects of acceleration of the early stages of rotting and earlier limitation when the temperature is raised from 0 to 10 “C. Tuber resistance would cause the cessation of rotting in the variety Majestic after 8 days at room temperature, and the interaction of the two factors would give rise to the extreme variability shown by the other varieties at this temperature. It is tempting to suppose that the corky appearance
Temperature
effects
on gangrene
development
485
which rots generally show at the higher temperatures is evidence of a resistance mechanism but there is no direct evidence to support this. It was generally found, as in earlier studies [5], that rots extend radially through the cortical tissues less than they do in depth into the medulla tissues. For example in the humidity experiment averaging over humidities, temperatures and varieties, the mean radial extension was 1 l-61 mm whilst the mean depth was 20.07 mm. It was also observed that rots extend symmetrically only at low temperatures and tend to follow the path of pith tissue at higher. This observation is evidence for differential tissue resistance and since it is more pronounced at higher temperatures is most likely not brought about by differences in nutrient status. As noted earlier, however, the possibility that temperature influences the availability of substrate cannot be ignored. Kranz [3] also concluded that the inner tissues were more susceptible to fungal growth. The mechanism of wound healing has been proposed to limit gangrene disease in potatoes (for example references [S, S]), and the temperature relations of this process [I] are similar to those proposed for the resistance mechanism in this paper. However, Kranz [3] has demonstrated that although wound healing will render wounded surfaces resistant to infection by the pathogen, it is unlikely to be effective in limiting fungal growth through the tuber tissues. He suggested that fungal growth is limited by another, as yet unknown, mechanism which is localized in the cortical tissues. It is thus possible that both mechanisms are operative on the farm and, if so, extreme care should be exercised in relating the laboratory findings to farming practice since in the laboratory the use of agar disks with growing mycelium and covering all inoculated wounds with “Sellotape” imposes conditions such that the pathogen always effects initial penetration and the observed resistance is that which limits fungal extension. Laboratory inoculation does, however, give reactions which correlate with susceptibilities found in agricultural practice [5]. Temperature and humidity will, no doubt, influence the process of infection but “Sellotape” ensures high humidity conditions in all cases. The acceleration of the early stages of rotting with rising temperature over the range 0 to 10 “C shown in all experiments would explain larger rots being found on tubers at 10 “C than at 5 “C when scored soon after inoculation. The tendency for limitation to be shown soonest at the higher temperature, however, means that with increasing length of incubation there would be an increasing possibility that rots would be larger at 5 “C than at 10 “C. This effect may explain the conflicting temperature relations reported in the literature since Langton [5] scored rots 13 and 21 days after inoculation and found growth more rapid at 10 “C, whilst Malcolmson [8] and Khan [Z] scored much later, after 42 days, and found the largest rots at 5 “C. Kranz [4], however, who also scored after 42 days, frequently found the largest rots at 10 “C. It has generally been reported that rotting decreases with increasing temperature over the range 5 to 20 “C, when scoring is after 42 days [Z, 4, 81. This relationship was shown by Majestic in the March sampling experiment in the present study but not by the other varieties. It is probable that many other factors, including time of testing, will modify the temperature effects. Thus, for example, the comparison between the results of the November and March sampling experiments will be influenced by the storage
486
F. A. Langton
conditions of the tubers before the samplings. The seasonal influence experiments showed that at both 5 and 10 “C rots became larger as the season advanced which is evidence of a progressive decline of tuber resistance (see also references [3, 8, lo]). The fall was particularly marked when testing was at 10 “C and at both temperatures was most pronounced between July and September. The fall is related possibly to the differential effects found; in July, when there was probably a high level of resistance, rots were largest at 5 “C, at which temperature resistance is poorly expressed, but later (February) when there might have been less resistance, rots were largest at 10 “C, the temperature favouring fungal growth. However, King Edward tubers in the sampling experiments gave results which cannot be explained on the basis of this relationship since, at 10 “C in March, smaller rots were produced after 28 days than in the previous November. This discrepancy remains unexplained. The five variety experiment gave disease susceptibility ratings in fair agreement with farm experience at both 5 and 10 “C with Arran Consul resistant, Ulster Chieftain susceptible, and the remaining three varieties reacting similarly in an intermediate position. Earlier work [5] has shown, however, that for acceptable varietal differentiation greater replication is required and better agreement might have been achieved in the experiment reported here had more tubers been used. Extensive use of this test method has shown, however, that King Edward VII does not react consistently as more resistant than Majestic although usually it appears so. Arran Consul does however, consistently appear resistant and Ulster Chieftain susceptible. It is, however, to be expected that varietal susceptibility in agricultural practice will be influenced by factors other than fungal extension through tuber tissues. Such factors may include susceptibility of tubers to damage and the ease with which the fungus can infect tubers. Varietal differences agreeing with those found in agricultural practice are shown more accurately in tests at 10 “C rather than at 5 “C [S]. The humidity experiment showed that high temperatures (20 “C) can give results in very poor agreement with farm experience. In the variety experiment variation increased with rot size to such an extent that although varietal means became more widely separated, varietal differences were no more sensitively detected with time once the 7 day stage had been passed at 10 “C and the 18 day stage at 5 “C. Incubation periods of about 28 days are used in varietal tests, but it appears that this could be reduced with no loss of effectiveness.
REFERENCES 1. ARTSCHWAGER,
E. (1927).
Wound
periderm formation
in the potato as affected by temperature.
3. ugric. Res. 35, 995-1000.
2. KHAN,
A. A. (1967). A study of some factors affecting infection and development of potato gangrene. Rec. agric. Res. Minist. Agtic. Nth. Ire. 16, 97-101. 3. KRANZ, J. (1958). Untersuchungen tiber die Phoma-Ftiule der Kartoffelknolle unter besonderer Berticksichtigung des Wirt-Parasit-Verhaltnisses. Phytopath. <. 33, 153-196. 4. KRANZ, J. (1959). tfber die Ausbreitung der Phoma-F&de der Kartoffelknolle in Abhingigkeit von Umweltfaktoren. Phytopath. <. 36, 101-l 10. 5. LANGTON, F. A. (1971). The development of a laboratory test for assessing potato varietal susceptibility to gangrene caused by Phoma exigua var. foveata. Potato Res. 14,29-38. 6. LOGAN, C. (1967). Potato gangrene. 3ournal of the National Association of Seed Potato Merchants
7, 78-80.
Temperature
effects
on gangrene
development
487
7. LOGAN, C. & KHAN, A. A. (1969). Comparative studies of Phoma spp. associated with potato gangrene. in Northern Ireland. Trans. Br. mycol. Sot. 52, 9-17. 8. MALCOLMSON, .I. F. (1958). Some factors affecting the occurrence and development in potatoes of gangrene caused by’ Phoma solanicola Prill. &-Delacr. Ann. a&l. Biol. 46, 639-650. 9. MALCOLMSON. 1. F. & GRAY, E. G. (1968). The incidence of eanerene of notatoes caused bv Phoma e.@$ in relation to handling and storage. Ann. appl. I&1.-62, 89-161. 10. TODD, J. M. & ADAM, J. W. (1967). Potato gangrene: some interconnected sources and factors. Proceedings of the 4th British Insecticide and Fungicide Conference 1967, pp. 276-284.