JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND Maataloustieteellinen Aikakauskirja Vol. 60; 159—178, 1988 Damping-off of sugar beet with special reference to the fungus Pythium Pringsheim Abstract. In Finland damping-off of sugar beet can be divided into two distinct phases. The first phase begins with the germination of the seeds and continues until the first true leaves have developed. Under field conditions seedlings usually remain healthy up to about 1 week after emergence. Thereafter a sudden outbreak of damping-off may occur, resulting in rapid wilting and death of seedlings. During the second phase of the disease, when seedlings have one or more pairs of true leaves, disease does not always result in the death of the plant; plants may survive throughout the summer. At the pernicious phaseof the disease the soil borne pathogen,Pythium debaryanum auct. non Hesse, is the most common causal agent, accounting in 1979—86 for 53.9 % (variation between years 18.3—90.1 %) of fungal isolations, and Fusarium species for28.3 % (5.0—58.5 %). At seedling stages with one or more pairs of true leaves Fusarium spp. predominate ac- counting for 49.4 % (36.1 —81.0 %) as compared to 23,9 % (2.9 —37.8) for P. debaryanum. The importance of Fusarium species as true damping-offpathogens is, however, doubtful. The seed borne damping-offpathogen Phoma belae Frank was isolated only in 0 to 4 % and was not dependent on the stage of seedling development. Of the factors affecting damping-off,high temperatures were repeatedly shown to increase the disease. This, presumably was an effect especially on P. debaryanum, the aggressiveness of which is strongly increased at high temperatures. Pot experiments showed preceding crops of cereals to have the best disease-decreasing ef- fect, both short-term (one growing period of preceding crop) and long-term (several growing periods of preceding crop) effect. Legumes kept the level of damping-off unchangedor even raised it, especially as a short-term effect. The influence of preceding crops varied in different soil types. Preceding crops also caused considerable fluctuations in inoculum density (0 to 3650 propagules/gram soil) and potential (0.2 —16 IPU M/gram soil) of Pythium. The correlation to damping-off of sugar beet was, however, poor. Seed treatment with the systemic fungicide hymexazol, especially when combined with thiram, prevented satisfactorily the pernicious type of damping-off. In many experiments this seed treat- ment repeatedly decreased disease incidence significantly, produced denser stands (7100—31200 numbers of beets more/hectare) and increased yield by s—lo5 —10 % on average. Index words; Damping-off sugar beet Pythium 159 https://www.c-info.fi/en/info/?token=uqUnSNlys20rRtoP.etxiV3u_b9TDBQDtpUXaDQ.iM3f2MeZTmJPDdCqIWZY3hZcJLNDf4ekVDcpiBCo3QfmAQYIe8kexKyX4nIlrrcrU5bPRKLR8PwAcPodmSVFxIVNddZ8Zw6UiDXb3begpj7zIjUuCbOl4R4G50tfTrlBDH2fmvFyYsqGieprZRHxukHCM2esPA 160 List of original articles The original articles summarized here are: I. Vestberg, M., Tahvonen, R., Raininko, K. & Nuormala, N. 1982. Damping-off of sugar beet in Finland. I. Causal agents and some factors affecting the dis- ease. J. Sci. Agric. Soc. Fin. 54: 225—244. 11. Vestberg, M., Tahvonen, R., Raininko, K. & Nuormala, N. 1983. Damping-off of sugar beet in Finland. 11. Disease control. J. Sci. Agric. Soc. Fin. 55: 431 —450. 111. Vestberg, M. 1984. Damping-off of sugar beet in Finland. 111. Effect of tem- perature and disease forecasting. J. Agric. Sci. Fin. 56; 283—290. IV. Vestberg, M. 1985. Experiments on direct isolation of Pythium spp. from Finn- ish sugar beet soils. J. Agric. Sci. Fin. 57:223—230. V. Vestberg, M. 1987. The effect of preceding crops on damping-off of sugar beet and some ecological properties of the fungus Pythium Pringsh. J. Agric. Sci. Fin. 59: 87—100. Reference to these publications is made in the text by citing the appropriate Roman numerals. Introduction Damping-off is defined as the collapse and falling over of young seedlings and herbaceous cuttings caused by the attack of various or- ganisms at or below soil level (Mc Kay 1952). In Europe in 1968—1970, the greatest losses due to damping-off of sugar beet occurred in Poland, Romania, Czechoslovakia, Hungary and Ireland (Dunning 1972). In 1979—1980, the main disease causing factors at the seedling stage in 16 European countries were in the decreasing order of frequency: Phoma (13), Pythiurn (13), Aphanomyces (8), Rhizoctonia (6), Fusarium (3) and Alternaria (2) (Dun- ning and Heijbroek 1981). Damping-off investigations from the Nor- dic countries are relatively few. In 1945, Björling published a very valuable work on Phoma belae. In Finland, Linnasalmi (1952) studied damping-off of a number of vegeta- bles and ornamental plants. Other investiga- tions on damping-off of sugar beet have been done by e.g. Möllerström and Klinteberg (1964), Rasmussen (1967), Linnasalmi (1970) and Möllerström (1974). Damping-off has since long been a well- known disease among Finnish sugar beet growers. Earlier, when using the normal diploid multigerm beet seed, the amount of seeds used for sowing was great and growers could afford to lose seedlings because of damping-off. In the late 60s a new type of su- gar beet seed, genetic monogerm seed, was in- troduced in Finland. This made it possible to rationalize the cultivation technique by sow- ing into stands of final density. Now the grow- ers could no longer afford to lose seedlings. Hence the importance of damping-off of su- gar beet increased and has been estimated to be the main reason for thin brairds in the 70s. Yearly 150—300 hectares of almost complete- ly destroyed beet stands have been resown. In 1979—1980 with heavy attacks of damping- off the total yield of Finnish sugar beets was estimated to decrease by about 8 % as a result of damping-off (Raininko and Vestberg 1981). The aim of this study was to identify the causal agents of the disease, to study factors affecting the disease and to find ways to con- trol it. Special attention was paid to the fun- gus Pylhium and its role in the damping-off disease. Results and discussion 1. Disease symptoms In Finland, damping-off of sugar beet can be divided into two distinct phases (1) similar to those described by Warren (1948). First, there is the very severe phase, which rapidly results in the death of the seedlings. This phase begins with the germination of the seed and continues until the development of the first true leaves. Warren (1948) further even divided this phase into a pre-emergent and a post-emergent phase. In Finland, pre- emergence damping-off occurs only during some years and at some localities. This type of damping-off can be observed as gaps in the braird at the time of emergence. Usually, however, seedlings emerge quite well and the first disease symptoms appear only 7—lo days after emergence. Infection at the cotyledon stage soon after emergence usually leads to complete wilting and rapid death of the plant. A water-soaked, brown to grey or black area extends up and down the hypocotyl or the upper portion of the young taproot from the point of entry of the pathogenic organism. Discoloration may also, in later stages extend up into the peti- 161 oles of the cotyledons. The collapse of the hypocotyl of the seedling is followed by desic- cation. Under favourable conditions this de- velopment may take place within I—21 —2 days (I)- The second phase of the disease is charac- terized by the seedlings not being immediate- ly killed; they may stay alive for quite a long time. Such diseased seedlings have a dark thread-like root. The disease is, however, re- stricted to the cortex, which explains why the seedlings do not die. If the disease advances to the vascular bundle of the root, the seedling will die (Butler& Jones 1961). However, the neck collar is weak and typically constricted thus, later in the summer they easily break at the root collar due to strong winds or agricul- tural proceedings. In the autumn at harvest- ing the abnormally developed beets can easi- ly be separated from the healthy ones. It was shown that the weather conditions in July and August determine how well seedlings will recover from the second phase of damping- off (I). In summers with high temperatures and sufficient rainfall the recovery is better than in cool summers. Chronic damping-off, which appear? after the true damping-off phase, is sometimes also named “strangles”, which refers to symptoms caused by abiotic factors such as strong winds or dry soil (Boyd 1966, Schollmeyer 1980). Acid soils are also reported to cause damping-off-like injuries in beet seedlings (Gates and Hull 1954). Under Finnish conditions, however, it seems clear that damping-off of sugar beet is mainly caused by microorganisms and not by abiotic factors (1). Damping-off of sugar beet usually appears in the same field during several years in suc- cession and theappearance is clearly patchy. Sometimes the disease proceeds along the row, not from one row to another. In this study no attempts were made to identify causing organisms on a macroscopic level. Such an identification is difficult to make because the symptoms caused by differ- ent species of microorganisms do not differ much from each other (Coons and Stewart 1927, Benada et al. 1987). In many cases there are also mixed infections (Coons and Stewart 1927). However, Coons and Stewart (1927) and Mc Kay (1952) divided the symptoms into three groups, which can be connected to certain damping-offpathogens: 1) Phoma betae: The affected hypocotyl turns brownish to black. The lesions are dry and the attack is chiefly confined to the cor- tex of the stem. The destruction of the seed- ling is rather slow. 2) Pythium and Aphanomyces: There is often a rapid wilting of seedlings. The lesions are soft and water-soaked. 3) Rhizoctonia solani: This fungus causes rather dry, brown lesions, which spread slow- ly. The taproot is often decayed, which initi- ates the development of rootlets above the decayed region. 2. Causal agents Introductory experiments to study the causal agents of damping-off on sugar beet were carried out in a glasshouse by studying the damping-off microflora of 48 soil samples from different parts of the Finnish sugar beet growing district (I). A fungus of the genus Pythium was found very frequently. Accord- ing to the key of Waterhouse (1967), the spe- cies was named P. debaryanum auct. non Hesse. Just after emergence P. debaryanum accounted for about 95 % of the total fungal isolates and 35 days after emergence still for 78 %. Correspondingly, other damping-off pathogens occurred sparcely, Fusarium spp. averaged 2.8 %, Phoma betae 2.2 % and Rhizoctonia solani 0.4 °Io. The damping-off flora of sugar beet seed- lings grown in the field was studied in 1979—86 (Table 1). A total of about 8400 seedlings were studied. Study I presents the results of the years, 1979—1981. Diseased seedlings were collected at the cotyledon stage, 7—lo days after emergence. A second collec- tion was made about 20 days after emergence. Similarly to the pot experiment, P. debarya- 162 163 num was the most common fungus, especial- ly at the cotyledon stage, accounting for 53.9 % of the fungal isolations. Under field con- ditions the fungus was not, however, so dominating as it was in the pot experiment. Variations between different years were also considerable, 18.3—90 °7o in 1979—1986, which indicates the importance of climatolog- ical factors. The results of this study are in agreement with those of Buchholtz (1938), Nolle (1960), Peshel(l969) and Kuhnel(l97B) ac- cording to which the most widely distributed damping-offpathogens on sugar beet belong to the genus Pythium. Within the genus, the species P. debaryanum Hesse, P. ultimum Trow and P. aphanidermatum (Edson) Fizp. are the most commonly isolated (Buchholtz 1938, Hills and Leach 1952, Gatesand Hull 1954, Till 1968, Linnasalmi 1970, Böttcher and Behr 1980). P. aphanidermatum is found especially in the United States (Till 1968, Takahashicl al. 1972). Other isolated species of Pythium include P. irregulare Drechs. (Vesely 1978, Böttcher and Behr 1980), P. spinosum Sawada apud Sawada & Chen (Takahashi et al. 1972), P. mamillatum Meurs (Meurs 1928), P. elongatum Matth., Table 1. Frequency of damping-off pathogens on sugar beet in 1979—86: a) about 7—lo days after emergence (3900 seedlings studied) and b) about 20 days after emer- gence (4500 seedlings studied). P. paroecandrum Drechs., P. echinulatum Matth. and P. roslralum Bull. (Böttcher and Behr 1980). The actual position of the Pythium species found most commonly in this investigation is not clear. According to the latest taxonomic studies on Pythium, there exists no species named P. debaryanum (Van Der Plaats- Niterink 1981). Isolations of P. debaryanum have in fact proved to be misidentifications of either P. ultimum, P. intermedium, P. ir- regulare or even of P. sylvaticum. In this study, however, the species of Pythium found very commonly in Finnish sugar beet soils is named P. debaryanum after Waterhouse (1967). At the second collection, Fusarium species were the most frequently isolated. Of all the fungal isolations made, they accounted for 49.4 % as compared to 23.9 % for P. de- baryanum. Out of seven species of Fusarium isolated, F. culmorum (W.G. Sm.) Sacc., F. oxysporum Schlecht. and F. sambucinum Fuck, were predominating. All these species are common soil saprophytes (Domsch et al. 1980), and their actual role as damping-off pathogens is not established. According to Hodges (1936), Möllerström and Klinteberg (1964) and BorrcHEßand Behr(1980), Fusar- ium species can be considered primary patho- gens on sugar beet seedlings. Gatesand Hull (1954) found the species of Fusarium to be weak damping-off pathogens in acid soils (pH < 6.5). In Finnish mineral sugar beet soils pH is about 6.3 (Pelo 1987), which would indicate pathogenicity ofFusarium as a damping-offpathogen. However, in pathoge- nicity tests, three Fusarium species caused no disease when inoculated into peat substrate. P. debaryanum and P. betae, on the contrary, caused disease symptoms in beet seedlings un- der the same experimental conditions (I). In extensive pathogenicity experiments Lin- nasalmi (1952) also found Fusarium species to be only slightly or not at all pathogenic as causal agents of damping-off on cabbage, cauliflower, cucumber and tomato. Vesely (1976) isolated 10 species of Fusarium from Pathogen Isolated pathogens % of all fungal isolations Average Yearly 1979—86 variation a) 7—lo days after emergence Fusahum spp. 28.3 5.0—58.5 Phoma belae 1.2 0—4.0 Pythium debaryanum 53.9 18.3—90.1 Khizoelonia solani 0.2 0—3.1 b) 20 days after emergence Fusahum spp. 49.4 36.1—81.0 Phoma betae 1.4 0—3.3 Pythium debaryanum 23.9 2.9—37.8 Rhizoctonia solani 0.2 o—o.B diseased sugar beet seedlings. Although all species were only slightly pathogenic, they caused excessive root branching in emerging seedlings. Isolations of P. betae varied between 0 and 4.0 % according to sampling time and year (Table 1). The general occurrence of this fun- gus was not dependent on the stage of seedling development. According to many investiga- tions, P. betae is an equally important damping-off pathogen as Pythium on sugar beet (Mc Kay 1952, Nolle 1960, Möller- ström 1964, Leach and Macdonald 1976). Linnasalmi (1970), in an earlier Finnish in- vestigation also found P. betae to be the most common fungus on diseased sugar beet seed- lings, followed by Pythium debaryanum and Fusarium spp. . Because P. betae is a seed borne pathogen and can hardly overwinter in the soil (Pool and Mc Kay 1915, Me Kay 1952), the reason for the decline as a causal agent of damping-off in Finland must be that the sugar beet seeds are quite free from infec- tion. The fungi Alternaria alternata and Ulocladium consortiale (Thiim)Simm were isolated rather frequently in some years (I), but the pathogenicity of these fungi is mostly weak (Vesely 1977b), although contradicto- ry opinions also exist (Greis 1940, Heidel and Schultze 1984). In this study, the damping- off pathogen Rhizoctonia solani was found only in a few samples. Generally, species of Rhizoctonia, i.e. R. solani (Coons and Stewart 1927, Hills and Leach 1952) and R. violacea(Tul.)Pat (Afanasiev and Morris 1942, Benada et al. 1987), have not been found to be as important pathogens on sugar beet as Pylhium and Phoma betae. Species of the soil borne fungus Aphanomyces commonly give rise to damping-off on sugar beet both in Europe (Schäufele and Winner 1972, Byford and Stamps 1975) and the United States (Coons et al. 1948, Papavizas and Ayers 1976). However, in this study, no species of Aphanomyces were found. As can be decided from their occurrence, only fungi belonging to the genera Pythium and Fusarium have any significance as damping-offpathogens on su- gar beet in Finland. 3. Factors affecting the disease Björling pointed out already in 1945 that beet damping-off is a pronounced predispo- sition disease, which is affected by different abiotic and biotic factors like soil and climat- ic conditions. In practice, this appears as var- iations in the occurrence of damping-off be- tween years and fields and even within fields. Whithin fields the damping-off shows a typi- cal patchy appearance, the reason for which is not fully understood (Buchholtz 1938). 3.1. Abiotic factors 3.1.1. Soil type There are diverging opinions about the role of soil type in damping-off of sugar beet. Ac- cording to Gram (1927), the physical condi- tions of the soil are more important than the soil type. Coons and Stewart (1927) agree with this, but at the same time they claim that the most severe outbreaks of damping-off ap- pear on heavy soils and highly organic soils. However, contrary to this, Gatesand Hull (1954) found lower incidence of damping-off in clay soils than in light soils. Remy (1950) found damping-off in all kinds of soils, but more frequently in clay than in sand. Ur- banovich(1965) found a correlation between the humus content of the soil and damping- off incidence. Angell (1954) isolated the damping-off pathogen Pylhium mainly in the uppermost soil layers and in coarser soil types. According to Likais(l94B), the aggressiveness of P. debaryanum is related to the colloid con- tent of the soil. At lower colloid contents the aggressiveness will decrease. On the other hand, Nolle (1960) found no differences in the agressiveness of Pylhium inoculated into compost or mineral soil. In this investigation, the content of humus or clay was determined in 47 soil samples from 164 heavily infested sugar beet fields (I, Table 4). The humus content variedbetween 1.7 % and 44.3 % and the clay content correspondingly between 2 % and 73 %. However, no corre- lations were found between these figures and the incidence of damping-off. These results are in agreement with those of Gram (1927) and Buchholtz (1938). 3.1.2. Soil acidity Many authors have found a correlation be- tween pH and the severity of damping-off of sugar beet. Arrhenius (1924) claims that rais- ing the pH to 7.2—7.6 by the use of lime will contribute to better control of Pythium damping-off. Other investigations show simi- lar associations between pH and damping-off (Mc Kay 1952, Griffin 1958). On the other hand, damping-off of sugar beet caused by the seed-borne pathogen Phoma betae is not de- pendent on soil acidity. It may occur also in strongly alkaline soils (Arrhenius 1924, Me Kay 1952). In this study, the pH values of 47 soil sam- ples from infested sugar beet fields varied be- tween 5.1 and 6.9 (mean 6.2). No correlation, was noticed between pH and damping-off (I, Table 4). In a pot experiment, the raising of pH from 5.2 to 6.6 by the use of lime had a significant disease decreasing effect (11, Table 5). The effect of liming and higher pH levels on damping-off of sugar beet was studied un- der field conditions during two years (11, Ta- bles 15 and 16). Only a very slight disease decreasing effect was noticed. In one experi- ment, liming decreased the sugar content of the beetroots (11, Table 16). The acidity of Finnish sugar beet soils is often quite pronounced. Therefore it is not in practice possible by liming to raise the pH values of the fields to 7.2—7.6 which would control damping-off, as suggested by Arrhenius (1924). On the other hand, Buchholtz (l93B) found good mycelial growth of Pythium with- in the pH values 5 and 7.5, which indicates that Pythium damping-off could be severe even on alkaline soils. Calcium, a component of lime, on the other hand, has been shown to stimulate production of oospores of Pythi- um in vitro, which would increase the damping-off potential of the soil (Haskins 1965, Lumsden and Ayers 1975). 3.1.3. Soil nutrients Generally, a balanced mineral level in the soil provides plants the best possibilities to prevent attacks by damping-off. Phosphorus is reported to have a preventive effect against damping-off (Afanasiev and Carlson 1943, Möllerström and Klinteberg 1964). However, in this investigation no correlation was observed between the level of phospho- rus and damping-off in sugar beet (I, Table 4). The amount of potassium, sodium or mag- nesium in the soils studied correlated slightly to damping-off, which is in agreement with Yale and Vaughan (1962). Manure has also shown a preventive effect against damping-off (Afanasiev and Carlson 1943, Young 1943). 3.1.4. Moisture Soil moisture is an important concept de- termining germination and growth of microor- ganisms. The frequency of, for example, bac- teria occurring on buried slides or within pota- to lenticles decreases rapidly below field ca- pacity. The clamydospores of Fusarium cul- morum, which germinate in very dry soil, even at soil water potentials down to -85 bar are another extreme. Pythium spp. and Mortierel- la, on the other hand, colonized buried plant parts most frequently in relatively wet soils at soil-water potentials exceeding -1 bar. (Griffin 1972). Buchholtz (1938) found no significant differences in soil moisture between healthy and diseased areas within sugar beet fields. Several authors (Roth and Riker 1943, Zhukova 1953, Barton 1958) report an in- crease in aggressiveness of Pythium damping- off close to water saturation. According to Doran (1946) and Zhukova (1953), an in- crease will be observed in Pythium damping- off when soil moisture exceeds 65 %. 165 In Finland, soil moisture is usually high af- ter the melting of snow in spring, during the initial development of the sugar beet seedlings (Brummer 1960). This is a prerequisite for a rapid infection of the seedlings by Pythium. At high soil moistures the thick cell walls of dormant Pythium oospores become thin. Dur- ing germination such oospores readily infect hypocotyls of young seedlings (Hoppe 1966, LuMSDENand Ayers 1975). High soil moisture is also a prerequisite of saprophytic growth of Pythium in soil (Hendrix and Campbell 1973). Later on, in June, dry periods with quite high temperatures are not rare. This might contribute to the common occurrence of Fusarium species in damping-off diseased beet seedlings at this time (I). It has been shown that Fusarium species are favoured by low soil moisture, even down to 30 % of satu- ration (Shen 1940). 3.1.5. Temperature Of all the physical variables of biological significance, temperature is perhaps the most obvious. Even during one day, a fluctuation of 35°C may occur at the soil surface in tem- perate zones (Russell 1961). With increasing depth, the fluctuations of diurnal temperature are reduced and approach a mean value. This might be only 10°C in summer in temperate climates (Griffin 1972). In Denmark, Mikkelsen (1982) tried to compare the general occurrence of damping- off with temperature during May—June in 1954—82. He found heavy outbreaks in warm early summers, while the disease remained at a low level in cool summers. This could be no- ticedalso in Finland when comparing average temperatures in the early summers of 1979—87 (Helminen 1979—1987) with aver- age damping-offpercentages in sugar beet ex- periments during these years. This relation could, however, not be statistically verified. The effects of high temperatures on the in- crease of damping-off was demonstrated in a pot experiment at B°C and 18°C (1, Table 2). Damping-off averaged 26.7 % and 54.1 % respectively. In practice there is often a very rapid out- break of damping-off following a period of high temperatures. Periods of 20—25°C daily temperatures are not rare during the ear- ly seedling stage of sugar beet in late May or in June. By the use of climate chambers the effect of periods of high or low temperatures before or after emergence on the severity of damping-off was studied. Two highly infest- ed sugar beet soils were used (III). A constant low temperature (15°C day and B°C night) gave rise to an average damping-off of 45 % as compared to 97 % at constantly high tem- perature (25°C day and 15°C night) (111, Table 1). When the pots were kept at low tem- perature up to emergence and at high temper- ature thereafter, the degree of damping-off was the same as in seedlings grown at continu- ously high temperatures. The opposite situa- tion gave a damping-off incidence of 62 % which indicates that damping-off had started during the pre-germination phase. At least a 7-day period of high temperature was needed to cause an increase of disease if the pots had been held at low temperature up to emergence. In the peat soil, at 14 day’s period of high tem- peratures was needed to increase disease sig- nificantly. The length of the warm period cor- related significantly with the degree of damping-off (r =0.938*** and o.Boo***, respectively) (III). When evaluating the role of temperature on damping-off, the temperature requirements of different causal agents should be taken into consideration. The optimum in vitro growth rate of Pythium debaryanum is observed at 25 —30°C (Buchholtz 1938, Middleton 1943). Under natural conditions, in the soil, the optimum occurs at somewhat lower tem- peratures due to competition from antagonists at the higher temperature (Sverrisson 1979, Lifshitz and Hancock 1983). Phoma betae has a somewhat lower optimum than P. de- baryanunt. Björling (1945) found an opti- mum germination of pycnospores of P. betae at 20—25 °C and some germination even at O°C. In practice, damping-off of sugar beet caused by P. betae is favoured by low soil tem- 166 peratures (Jacks 1951, Gates and Hull 1954, Nolle 1960). Comparing the growth of P. betae and P. debaryanum, Nolle (1960) found a tenfold faster growth per °C of the latter at s—l2°C. The species Fusarium cul- morum, F. oxysporum and F. sambucinum, found most frequently in this study (I), all have optimum in vitro growth at 25—30°C (Domsch et al. 1980). In Finland, the soil temperature at and af- ter sugar beet seedling emergence is about 10—12°C at a depth of 20 cm (Helminen 1979—1987) and exceeds that greatly at the soil surface. This favours Pythium damping- off and Fusarium fungi as well. Buchholtz (1938) and Nolle (1960) consider + 15°C a critical point, above which the aggressiveness of Pythium damping-off increases rapidly. Temperature also affects the pre-emergence/ post-emergence damping-off ratio (I, Greeves and Muskett 1936, Buchholtz 1938). A high temperature gives good emer- gence, but emergence is closely followed by an outbreak of disease. At low soil tempera- ture the germination is slow and proportion- ally poorer than at high temperature, but the final survival of seedlings is higher than at high temperature. Meteorological factors such as temperature and humidity act on the pathogen or on the host or on both when brought into associa- tion. Therefore it may often be difficult to de- cide how much of a disease increase is at- tributable to decreased seedling resistance to a pathogen and how much to increased aggres- siveness of the pathogen. Mikkelsen (1982) emphazises that beet seedlings grow very rapidly at high temperatures. This will result in elongated thin-walled cells, which are very sensitive to attacks of damping-off. 3.2. Biotic factors There are three basic ecological properties of soil borne fungi, which influence the dis- ease expression: inoculum density (ID), inocu- lum potential (IP) and competitive saprophyt- ic ability (CSA) (Bouhot 1979). These con- cepts are useful when studying, for example, environmental factors (temperature, soil moisture, C/N ratio, preceding crop, etc.) or the mechanisms of biological control (Baker 1971), or they may be used to forecast soil borne diseases. Introductory studies of ID and IP of Pythi- um were made in order to get a better picture of the variations in Pythium damping-off on sugar beet. 3.2.1. Inoculum density of Pythium 3.2.1.1. Concept Inoculum density is quantitatively measured as the number of propagules of a pathogen per gram of dry soil (Bouhot 1979). There are many investigations on the quantitative relationship between ID and soil borne dis- eases, with e.g. Pythium spp. (Mitchell 1978, Ferriss 1982), Rhizoctonia solani (Van Bruggen et al. 1986), Fusarium spp. (Guy and Baker 1979) and Phytophtora (Mitchell 1978). For direct quantitative isolation of Pythi- um from soil, Warcup’s (1950) soil plate method has proved superior to the dilution plate method. Schmitthenner (1962) used a soil-particle technique for isolation of P. ul- timum and several other Pythium species from soil particles. Stanghellini and Hancock (1970) found that P. ultimum grew out from small drops dispersed on the surface of 3-day- old water agar and that this method could be quantified by making dilutions. In the present study, Pythium species were successfully iso- lated directly from soil and the ID was mea- sured using the soil-plate method of Warcup (1950) as modified by Ricci et al. (1976). Ac- cording to this method, small amounts of oven-dried soil were evenly dispersed in 2.5 % water agar at 40—42°C. Citric acid, 50 ml/1, was added to the agar before autoclavation. After solidification of the agar, round discs, 1 cm in diameter, were cut out and transferred to the Pythium selective medium, Martin’s (Martin 1950) agar to which benomyl and PCNB had been added (15 ppm of each). The 167 agar plates were incubated for 24 h at 15°C in the dark, after which they were exposed to normal daylight and darkness. After four days, the number of plates with mycelia of Pythium was recorded and the number of propagules per gram of dry soil was calculated according to the MPN method (Maloy and Alexander 1958) (IV). 3.2.1.2. Applications The method yielded 4 types of soil borne Pythium species from sugar beet soils includ- ing the pathogenic type (IV, Fig. 1). However, this type could not be distinguished from the saprophytic ones without inoculation experi- ments with every strain (Bouhot 1979). The method proved good for estimating propagule densities of very heavily infested soils, but it is not useful to detect low oospore densities of, for instance, 1 propagule or less per gram of soil. In pot experiments propagules of Pythium numbered from near 0 to 3650 in naturally in- fested sugar beet field soil after different preceding crops (V, Fig. 1,2, Table 4). The ID of Pythium correlated, however, poorly with damping-off of sugar beet seedlings. Un- expectedly, in some cases a significant nega- tive correlation was observed between ID of Pythium and damping-off. On the whole, the results seem to support the view of Diamond and Horsfall (1965) who claim that, only in exceptional cases is inoculum density of a pathogen directly correlated with the disease. Many authors use transformations of dis- ease percentages to obtain better correlations with ID of a pathogen (Baker 1971, Ferriss 1982, Gilligan 1983). Bouhot and Joannes (1979) compared in a material of more than 600 soil samples four mathematical transfor- mations of disease percentages. In 80—90 %, the log-log and the probit-log transformations proved the best. In the present investigation there was no overall improvement of correla- tions between ID and disease by the use of transformations, although in some cases this did happen (V). The material is, however, too small to draw any conclusions in this respect. 3.2.2. Inoculum potential of Pythium 3.2.2.1. Concept According to Bouhot (1979), the system inoculum potential (IP) disease is the most appropriate for Pythium to calculate the risk of obtaining disease. The IP of a pathogen has been defined in various ways. According to Diamond and Horsfall (1965), it can be defined in a broad sense as the result of theaction of the environ- ment, the vigor of the pathogen to establish an infection, the susceptibility of the host and the amount of inoculum present. Martinson (1963) defines the term as a function of inocu- lum density or intensity, available nutrient and genetic capacity of the organism. According to Bouhot (1979), the number of successful infections obtained in optimum environmen- tal conditions on a standard susceptible host is in practice the only valid measure of IP. For reliable and replicable results in the estimation of IP, the following criteria must be met (Bouhot 1979): 1) Select an indicator plant susceptible to the parasite. 2) Use the plant at its most sensitive period. 3) Apply the naturally infested soil sample to the most sensitive part of the plant. 4) Standardize the environmental conditions so that the inoculum potential constantly induces maximum disease. 5) Quantify the techniques by progressively diluting the soil sample. 6) Determine optimal conditions for the highest selectivity, sensitivity and rapidity of the technique. The sensitivity of the bioassays can be in- creased by adding selective substrates to the soil to increase the mass of inoculum. Pythi- um spp., which are weak competitors against other microorganisms in soil (Hendrix and Campbell 1973), can readily colonize in- troduced organic baits like corn (Liu and 168 Vaughan 1965), papaya tissues (Trujillo and Hine 1965) or oats (Yarwood 1966). Oat meal increased the sensitivity of detection of Pythi- um by at least hundredfold (Bouhot 1975 a). Furthermore, a quantification factor can be introduced. Bouhot (1975 b) diluted the test soil with sterile soil and obtained a partial linear relationship between the dilution rate and the amount of disease in the indicator plants. He used the linear part of the cur- vilinear graph to calculate the IP of the soil. He calculated an inoculum potential unit (IPU SO ), which is defined as the minimum quantity (g) of test soil necessary to induce 50 % mortality in the plant population under standard experimental conditions. 3.2.2.2. Applications The IP of Pythium was determined at the end (growing periods 7 and 8) of a glasshouse crop rotation experiment (V, Fig. 3). The ex- periment was set up on three soil types, i.e. peat, very fine sand and sandy clay. The var- iation in numbers of IPU50/g soil between growing periods and rotations was consider- able, the values ranging from 0.2 to 16. In all three soil types, rotations with a high sequence of cereals exhibited on average lower IP of Pythium than did continuously cultivated su- gar beet which, however, had the highest IP in only one case. In peat and sandy clay rota- tions with the leguminous crop field bean caused great increases in the IP of Pythium. In the very fine sand, however, the highest IP were found in rotations with barley and grass (V, Fig. 3). Although ID and IP of Pythium are closely related ecological concepts they correlated poorly with each other (V, Table 7). To understand the variations in the results for ID and IP of Pythium and the mostly weak correlations between these and damping- off of Pythium, one must also take the con- cept of competitive saprophytic ability (CSA) into consideration, although it was not esti- mated in this investigation (V). CSA is defined as the ability of phytopathogenic fungi to de- velop saprophytically in the soil, which can ex- plain their multiplication and survival in the absence of a susceptible host. Furthermore, CSA may offer an explanation to the fluctu- ations in ID too (Bouhot 1979). For estima- tion of CSA the Cambridge method is fre- quently used (Butler 1953, Lukas 1955, Dhingra et al. 1976). According to this method, calibrated fragments of dead plant tissues are placed in the soil to trap the fun- gus to be studied. Cook (1970) buried fresh or autoclaved wheat straws to study saprophyt- ic colonization of Fusarium roseum f. sp. cerealis ‘culmorum’. Bouhot (1980) used a simplified version of Robertson’s (1975) paper disc method to measure the CSA of Pythium. Bouhot (1979), referring to several inves- tigations about ID and CSA in relation to dis- ease, concluded that disease severity in the case of Pythium spp. and Rhizoctonia solani is much more correlated with CSA than with ID. This, however, is less evident for root- inhabiting fungi like F. oxysporum, Verticil- Hum and Gaeumarmomyces. 3.2.2. Preceding crops Monocropping is common in Finnish sugar beet cultivation. This is thought to be one of the main reasons for the severe outbreaks of damping-off during the last decades. The effect on damping-off of different preceding crops as compared to sugar beet monocropping was studied in pot experiments in the glasshouse (V). Short-term effects, that is 4-month cultivation of a breaking crop, showed that legumes on average tended to keep the level of damping-off unchanged or even to raise it as compared to continuously cultivated sugar beet. Graminous plants, es- pecially cereals, on the other hand, had an op- posite short-term effect, increasing emergence and the numbers of surviving sugar beet seed- lings (V, Fig. 1). These effects of preceding crops are in agreement with Coons and Koti- la (1935), Deems and Young(1956) and Mum- foro(l96B). Arndt and 8ehr(1973) found no general relation between black leg and crop 169 rotation but infection by Pythium was, however, more harmful on plots with narrow rotations and high concentration of sugar beet. The long-term effects of preceding crops were studied in a glasshouse experiment of eight growing periods. Preceding crops to su- gar beet were field bean, barley and grass, only one crop in each rotation. The sequences of sugar beet varied between 14and 86 % (V, Table 2). The experiment lasted more than three years. During that time, the seasonal variations in climatological conditions like radiation, temperature and air humidity were considerable. At growing period 8, beet monocropping showed a mean (of three soils) post-emergence damping-off percentage of 34.8 % as compared to 14.0—28.0 % for the rotations. In contrast to the short-term effect, the legume field bean decreased disease sig- nificantly, as did barley and grass, too. Cal- culated from growing period 8, the correla- tion coefficient between percentage of sugar beet in rotation and percentage of post- emergence damping-off was significant (r = 0.841**). This experiment also indicated that response to preceding crops may vary ac- cording to soil type. The results indicate that the effect of preceding crop is much an effect on the patho- gen Pythium, especially the immediate short- term effect of the growing crop. Legumes, es- pecially pea, had a disease increasing effect. Pea is often attacked by Pythium spp. (Robertson 1973, Ruokola 1979), which are related to those attacking sugar beet, suggest- ing that pea roots and root exudates can serve as suitable nutrients or energy sources also for Pythium species on sugar beet. It is a well- known fact that root exudates affect myceli- al growth and oospore germination (Brown and Kennedy 1966, Kraft and Erwin 1967, Agnihotri and Vaartaja 1968) as well as zoospore movement (Royle and Hickman 1964, Spencer and Cooper 1967, Kraft and Erwin 1968, Chang Ho 1970) of Pythium spp. In the case of cereals as preceding crops, on the other hand, the composition of root exudates may be unfavourable for Pythium. The long-term effects of preceding crops during several growing periods seem to differ from the immediate effect (V). Presumably, there will be an effect over time on the saprophytic microflora, on the soil fauna and on other soil components. The results present- ed here are not directly comparable to field conditions, because the influence of winter, for example, cannot be taken into account in glasshouse experiments. 4. Disease forecasting Introductory experiments were carried out to study the possibilities of forecasting disease outbreaks in the field. Soil samples collected from sugar beet fields in the spring and au- tumn 1980 and 1981 were taken to a glass- house, where the percentage of damping-off was determined. This was then compared with the percentage of disease in the field (111, Fig. 2, Table 3). Soil collected from 36 fields in spring 1980 gave damping-off incidence of 21.8 °Jo and 32.5 % in the glasshouse at B°C and 18°C, respectively, while soil collected from the same fields in autumn 1980 gave disease incidences of 31.3 % and 61.6 %, respectively. The dis- ease incidence in the glasshouse usually cor- related rather poorly with that in the field, but was on average somewhat better for samples collected in autumn (especially in high glass- house temperature) than in spring (III). It was concluded that the possibilities of forecasting disease outbreaks in this way are rather limited, which supports the works of Bartels and Winner (1971) who studied the Pythium infection of beet side roots with a view of making prognosis of damage by methods similar to those used in this study. When the temperature factor is taken into ac- count, a negative prognosis using the follow- ing criteria, however, should be possible: Sugar beet seedlings are grown under glass at a high temperature in soil collected from the field in autumn. 170 If no disease occurs in the glasshouse, the risk of outbreaks in the field is minimal. If heavy outbreaks occur in the glasshouse, there is a high potential risk of severe at- tacks also in the field. However, the dis- ease may remain at a low level if condi- tions unfavourablefor development of the disease prevail. 5. Disease control In the control of damping-off of sugar beet one must distinguish between soil borne and seed borne damping-off. In controlling seed borne damping-off of sugar beet, seed dressings with mercurial com- pounds or thiram have been predominating (Gates and Hull 1954, Gates 1959, Nolle 1960, LtiDECKEand Winner 1963, Linnasalmi 1970, Möllerström 1974, Flori et al. 1985, Maude and Bambridge 1985). In England, Byford (1972) reported that Phoma betae damaged only few seedlings in commercial crops, although infection in sugar beet seed clusters were 20—62 % in 1958—1970. Steep- ing of the seeds in ethyl mercuric phosphate controlled the fungus. Maneb and mancozeb have also been reported to control seed borne damping-off of sugar beet (Darpoux et al. 1966, Koch 1979, Hrubesh and Wieser 1978). Soil borne damping-off of sugar beet is more difficult to control than the seed borne type. Despite this, treatment of seeds with fun- gicides such as iprodione, metalaxyl, hymex- azol (Dunning and Heijbroek 1981), fenaminosulf (Leach and Hills 1960, Schultze and Bohle 1976), propamocarb and phosetyl-Al (Jamart et al. 1983) also af- fects soil borne Pylhium damping-off. In Finland, the soil borne pathogens Pythi- um debaryanum and Fusarium spp. are predominating in damping-off of sugar beet, while Phoma betae plays a minor role (I). Pro- tective fungicides such as mercurial com- pounds or thiram had an insufficient effect on the soil borne damping-off (II). In the con- trol experiments, therefore, the main stress has been on testing fungicides efficient especially against Pylhium, which is the main microor- ganism causing seedling death at early stages. A range of fungicides were tested as seed dressors. Moreover, intensified control such as seed treatment combined with row spray- ing at seedling emergence or seed furrow ap- plication was also studied. Biological control agents were tested alongside with the chemi- cal compounds (II). The report in 1966 of carboxamide that moved systemically in a plant and suppressed fungal activity within the plant initiated the era of systemic fungicides (Fry 1982), which have created new possibilities for control of soil borne pathogens. In this study, the sys- temic fungicide propamocarb, which is effi- cient against Pylhium species (Anon, 1978) proved efficient against soil borne damping- off in pot experiments but usually not under field conditions (II). This may be a conse- quence of temperature or pH. In the pot ex- periment the effect of the fungicide was good at low temperature but negligible at high tem- perature. Kaars Sijpesteijn and coworkers (1974), working with prothiocarb, which is closely related to propamocarb, found a high fungitoxicity of prothiocarb at pH 7 but negligible at pH 5.3. With regard to the effect of temperature, two glasshouse experiments at B°C and 18°C showed the response to seed dressings to increase with decreasing temperature, a fact that is in agreement with the findings of Gates and Hull (1954). At B°C the treat- ment of seeds even with mercurial compounds was somewhat effective against soil borne damping-off, but at 18°C the effect was negligible. Under field conditions, how- ever, only hymexazol and especially hymexazol + thiram proved effective seed dressors (11, Tables 7—12), a finding which is in agreement with Koch (1979). Seed treat- I ment with these fungicides gave good protec- tion of the seedlings against damping-off up to about two weeks after emergence. There- after damping-off did occur, but the disease 171 was mostly of the chronic type and not of the dangerous type. Although hymexazol alone had a negligible effect on damping-off in some experiments (11, Tables 9 and 10), the combi- nation hymexazol + thiram had always a sig- nificant disease decreasing effect. Hymexazol increased in different experiments the final number of beet roots per hectare in autumn by 7100—31200. The seed treatment hymex- azol + thiram increased sugar beet yield in field experiments on average by s—lo5 —10 % (II). Hymexazol is reported to be effective against a range of soil borne diseases, espe- cially those caused by Pythium, Fusarium and Aphanomyces (Anon. 1971). Hymexazol is not only an effective fungicide against these fungi, but also a plant growth promotor (Kukalenko and Volodovich 1979). Both pot and field experiments showed higher amounts clearly to improve the effect of hymexazol. The optimum would be about 10 g a.i./kg seed combined with thiram 4 g. The use of thiram together with hymexazol improved the health of beet stands more than did hymex- azol alone (II). The same was observed by Hrubesh and Wieser (1978) who also recom- mend mancozeb in this respect. Naked sugar beet seed was used in most ex- periments in this study. However, two experi- ments (11, Tables 11 and 12) showed that dis- ease control may be even better by the use of pelleted seed, a fact also stressed by Panday and Agnihotri (1985). However, the use of the fungicides in pelleted seed gave smaller yield increases than did fungicidal treatment of naked seed. Some investigators mention certain soil-row spray treatments to be superior to standard seed treatments (Hills and Leach 1952, Gerhold 1956, Schultze and Bohle 1976, Linnasalmi 1970). In this study, spraying with hymexazol and thiram at seedling emer- gence in a 5 cm broad band using high amounts of water (up to 20000 1/net hectare) gave almost a 100 % control of the disease (11, Table 10). However, the efficient fungicidal concentration used, 0.5 %, means a total amount of 7 and 3 kg a.i. per hectare of hymexazol and thiram, respectively, amounts which are not economically profitable to use. Moreover, the favourable effect of such band- spraying on the yield of sugar beet in compar- ison with seed dressing alone was very small, except on some localities with extremely se- vere outbreaks of the disease. The application of the fungicide, using small amounts, to the seed furrow in connection with sowing, gave good results, but this method needs further technical development (11, Tables 3 and 14). Pythium oligandrum Drechs., originally described by Drechsler (1946), was tested as biological control agent. The fungus was ap- plied in a seed treatment in the form of a pow- der biopreparation containing oospores. Although effects of P. oligandrum compara- ble to those of fungicides such as thiram (Vesely 1978, 1979) or fenaminosulf (Martin and Hancock 1987) have been reported, no evident protective effect of the hyperparasite was found in this study (11, Table 4,9). In field experiments, P. oligandrum even lowered the yield of sugar beet by about 15 % as compared to yield from untreated sug- ar beet seeds. This would indicate that the fungus acted as a pathogen on sugar beet and not as a hyperparasite on P. debaryanum. Ac- cording to Vesely (1977 a), P. oligandrum is itself a weak facultative parasite on sugar beet seedlings, but the losses caused by it are in the order of a few percents. Vesely and Hejda- nek (1984) also point out the importance of a relatively low temperature from seed germi- nation to emergence and during the cotyledon stage to get the highest benefit of the bi- opreparation. Another species of Pythium, P. nunn Lif- shitz, Stanghellini & Baker, (Lifshitz et al. 1984 a) has also been used for biological con- trol of Pythium damping-off (Lifshitz et al. 1984 b, Paulitz and Baker 1987). The possi- ble role of different genera of bacteria in the biological control of Pythium has been dis- cussed as well (Broadbent et al. 1971, Nel- son et al. 1986, Elad and Chet 1987). 172 Rel'erences Afanasiev, M.M. & Morris, H.E. 1942. Control of seed- ling diseases of sugar beets in Montana. Phytopath. 32: 477—486. & Carlson, W.E. 1943. The relation of phosphorus and nitrogen ratio to the amount of seedling diseases of sugar beets. Proc. Amer. Soc. Sugar Beet Technol. 1942: 407—411. Agnihotri, V.P. & Vaartaja, O. 1968. Seed exudates from Pinus resinosa and their effects on growth and zoosporegermination of Pylhium aferlile. Can. J. Bot. 46: 1135—1141. Angei i , H.R. 1954. Partial segregation of bacteria and isolation of Pylhium from the coarser soil fractions. Austr. J. 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Denna rotbrandsundersökninginleddes 1979 som sam- projekt mellan institutionen för växtpatologi vid Helsing- fors universitet och Centralen för Sockerbetsodling i Bjär- nä. Frän och med är 1985 överflyttades projektets växt- patologiska undersökningar frän Helsingfors universitet tili Mellersta Finlands forskningsstation vid Lantbrukets forskningscentral, Laukas. Mälet för undersökningen har värit att identifiera sjuk- domsalstrarna, studera faktorer med inverkan pä sjuk- domen saml att finna vägar för bekämpning av sjukdo- men. Speciell vikt har värit fästad vid den viktigaste sjuk- domsalstraren, svampen Pylhium debaryanum. Rotbranden kan under finländska förhällanden inde- las i tvä faser, nämligen en fas dä plantorna dör och en kronisk fas. Den första fasen börjar i och med frögroning- en och fortsätter över uppkomststadiet fram tili slutet av hjärtbladsstadiet. Denna fas innefattar även underjor- disk rotbrand, vilken dock inte förekommer i nägon större utsträckning i Finland. Plantorna har vanligtvis god upp- komsl och sjukdomsangreppet sätter in ca 1 vecka efter uppkomsten. Plantorna erhäller dä en för sjukdomen ty- pisk insnörning av rothalsen. Ofta breder det skadade om- rädet även ut sig nerät tili roten och uppät tili hjärtbladens bas. Angripnaplantor vissnar snabbt ner och dör, t.o.m. inom I—2 dygn efter angreppets början. Den mark- levande svampen Pylhium debaryanum är den domine- rande rotbrandsalstraren pä delta stadium. Svampen ut- gjorde 53.9 % av samtliga rotbrandssvampar isolerade under ären 1979—1986. Den ärliga variationen var dock ansenlig, 18.3—90.1 %. Av övriga isolerade svampar pä tidigt plantstadium förekom arter av marklevandeFusar- ium i medeltal 28.3 %((5.58.5 %), medan den fröburna rotbrandspatogenenPhoma belae’s andel var endast 1.2 % (o—4 %). Dä betplantorna insjuknar pä örtbladsstadiet överlever de ofta sjukdomen trots att roten blir trädaktigl tunn och svart. Pä hösten vid betupptagningenkan försommarens rotbrand ännu märkas som missformade betor med ur- gröpningar pä rothalsen. Fusarium är de allmännast isole- rade svamparna pä kroniskt rotbrandsstadium. Deras an- del utgjorde 49.4 %((36.81.0 %) av samtliga isoler- ade svampar i förhällande tili 23.9 ak ((2. 37.8) för P. debaryanum. Phoma belae förekom i ungefär samma ul- sträckning som pä hjärtbladsstadiet. Ett mängfald av försök har visat att rotbrandsangrep- pen blir speciellt omfattande vid höga temperaturer. I praktiken kan man ofta märkä mycket kraftiga angrepp efter perioder av höga temperaturer under hjärlblads- stadiet eller tidigt örtbladsstadium. Delta är troligen en verkan riktad speciellt pä Pylhium-svampen, vilken har kraftigt ökad patogenitet vid höga temperaturer. Sockerbeta odlas i Finland ofta som monokultur, vilket anses vara en bidragande orsak tili den omfallande före- komsten av rotbrand. Olika förfrukters möjligtvis rot- brandsdecimerande inverkan studerades i kärlförsök. Sädesslagen visade sig ha sädan inverkan medan baljväxler som förfrukt höll rotbrandsnivän oförändrad eller höjde den, speciellt vid endast 1 växtperiodsavbrytandc odling. Förfrukternas inverkan varierade ocksä betydligt enligt jordart. Flera växtperioders förfruktsodling skiljde sig frän 1 växtperiods odling av förfrukt sätillvida att även baljväxterna uppvisade positiv inverkan. Stora variationer i antalet förökningsenheter per gram jord för svampen Pylhium kunde konstateras efter olika förfrukter. Kor- relationen med rotbrand var dock för det mesta svag. Betning av fröna med den systemiska fungiciden hymexazol och speciellt med hymexazol kombinerad med thiram gav tillräckligt skydd mot rotbrand under hjärt- bladsstadiet, dvs. under sjukdomens dödande fas. Efter delta angreps plantorna, men angreppen var relalivt lind- riga. Betningen ökade beständstätheten i olika försök med 7100—31200 plantor per hektar och gav i medeltal ca s—lo5 —10 % högre avkastning. Bandbesprutning av betorna strax efter uppkomst med hymexazol gav sä golt som full- ständigt skydd mot sjukdomen. Inverkan av en säden be- handling pä avkastningens storlek i jämförelse med en- bart fröbetning var dock obetydlig, med undantag av mycket kraftigt rotbrandsbesmittade lokaler. 178