Rhizoclonia solani damping-off on spring turnip rape and spring rape ( Brassica spp . ) in Finland

Damping-off occurred in 90 % of the spring oilseed rape fields surveyed in 1981—1982. On the average, 10 % of the plants were infected with damping-off. 6 % of the fields were severely infected (> 30 % of the plants affected) by damping-off and 38 % were uninfected or only slightly infected ( < 5% of the plants affected). The incidence of dampingoff was higher in those fields which had been under oilseed rape in earlier years. In a crop rotation experiment, the amount of damping-off increased from 2 % to 20 %, and finally to 38 %, depending upon whether turnip rape had been grown on the same part of the field once, twice or three times. Crops other than Cruciferous ones were grown for 1 or 3 years between the turnip rape crops. Rhizoclonia solani Kiitin was isolated from 76 % of the affected plants. The R. solani isolates produced severe damping-off on rape and turnip rape in pathogenicity tests. R. sotani isolates from barley, potato and lettuce brought about only mild cases of damping-off, or else only a decrease in the growth of the plants. Fusarium avenaceum Sacc. was the only other isolated fungus which was pathogenic.


Introduction
The cultivation of spring turnip rape (Brassica campestris L. ssp.oleifera) and spring rape ( Brassica napus L. var.oleifera ) increased sharply in Finland during the 1970'5.The area under these crops at the beginning of the 1980's has been around * New adress: Agricultural Research Centre, Depart- ment of Plant Pathology, SF-31600 JOKIOINEN, Finland 55 000-60 000 ha, spring turnip rape ac- counting for over 50 000 ha of this area.The yields of spring oilseed rape have remained stable under the varying growing conditions in Finland and oilseed rape has been con- sidered to be extremely suitable as a break crop in cereal monoculture (Köylijärvi 1982).
The pathogens affecting spring turnip rape and spring rape under Finnish conditions have been largely neglected.In Sweden and Index words; spring oilseed rape, damping-off, Rhizoclonia solani elsewhere in Europe, Leptosphaeria macu- lans (Desm.)Ces.& de Not, Sclerotina scle- rotiorum de Bary and Alternaria spp.are the most serious pathogens affecting oilseed rapes.In addition to these, damping-off and foot rot are also serious pathogens in Cana- da (Berkenkamp and Vaartnou 1972, Raw- linson and Muthyalu 1979, Kruger 1979,   Svensson 1982).In a long-term crop rotation experiment carried out by the Department of Plant Pathology, the University of Helsinki, damping-off started to increase strongly on turnip rape after the crop had been grown on the same area twice or three times.This led to the decision to determine whether the damping-off situation was the same with commercial crops of turnip rape and rape.
Material and methods

The crop rotation experiment
Spring turnip rape is included in two rotations in the crop rotation experiment being carried out by the Department of Plant Pathology at v iikki.
In one of the rotations, turnip rape is grown on the same block of the experiment every second year.Either barley or wheat are used as the preceding crop.In the other rota- tion, spring turnip rape is grown on the same site every fourth year, the order of the crops in the cycle being turnip rape barley/ wheat oats broad beans.
The experiment was established in 1977.During the period 1979-1982 the turnip rape variety was »Torch», and in 1979 the variety »Span» was used.
In 1979 a sample of 300 plant individual was analysed.Owing to the increase in the amount of damping-off, the number of analysed plants was increased during 1980-1982  to 1200 plants/year, i.e. 25 plants/sample plot.The analysis was carried out at the growth stage 2 (Harper and Berkenkamp   1975).
2. Collection of seedling samples from fields and their analysis Samples of spring turnip rape and spring rape seedlings were collected from 62 fields in southern Finland in 1981 and 50 fields in 1982.There were usually 2-4 true leaves on the seedlings at the time when they were collected.A single sample (about 400 seedlings) consisted of 4 subsamples which were taken from different points in the field.Each sub- sample consisted of about 100 seedlings which were collected from 2-4 adjacent rows.Soil samples (10 litres/field) were also taken from the topsoil in 1981.In addition, information about the cultivation history of each field during the preceding four years was collected in 1981.
The subsamples from each field were anal- ysed separately.The growth stage of the seedlings was determined and the seedlings classified into three groups: healthy = 0, infected = 1 and dead = 2.The damping-off °7o and disease index were calculated for each field as the mean of the subsamples.
Seedlings were grown in the soil samples at a high temperature ( + 25°C) and a low tem- perature (+ 11°C).Spring turnip rape of the »Torch» variety were used as the test plants.Plastic pots (1.5 1) were filled with the sample soil and 30 seeds sown in each at a depth of 2 cm.A pot containing sterilised sand instead of soil was used as a control.There were three replications.After the plants had grown for three weeks they were classified in the same way as the seedling samples taken from the fields.Only 30 soil samples could be studied at the low temperature.

Fungal isolations
Fungal isolations were made on plants suf- fering from damping-off.A small piece of tissue was cut from the borderline between healthy and infected tissue.The specimen was surface sterilised for 30 s in 1 % NaOCl and then rinsed in 94 % ethanol.When dry the specimens were transferred onto cornmeal agar (Difco) containing 100 ppm strepto-pension (1 Petri dish/100 ml sterile water) mycin (Streptomycin sulphate).The petri dishes were incubated at room temperature and the fungi which developed from the plant tissue were identified under a stereo and a light microscope after 7 days and after 21 days.

Pathogenicity tests
The pathogenicity of 16 fungal isolates to spring turnip rape and spring rape was tested in 1981.Seven out of the ten Rhizoctonia solani Kuhn isolates were from spring turnip rape or spring rape.The other 3 Rhizoctonia solani isolates were obtained from potato, barley and lettuce.
The Fusarium species included in the test were Fusarium avenaceum Sacc., Fusarium culmorum Sacc.Fusarium oxysporum Schlecht.and Fusarium sambucinum Fuckel.In addition, the pathogenicity of Ulocla- dium consortiale (Tiihm.)Simmons and Preussia aemulans (Rehm) v. Arx was tested.The latter fungus was isolated from each of the spring turnip rape seedlings suffering from damping-off which were growing on one field in Halikko.
The pathogenicity test was carried out both in a Jacobsen germinator and as a pot experiment in the greenhouse.The fungal isolates for the test were grown for one week on PDA medium (Difco) in 9-cm Petri dishes.
The test plants used in the Jacobsen germinator were spring turnip rape »Span».The seeds were immersed in an aqueous suspen- sion (1 petri dish/100 ml sterile water) of the test fungus.When the infected seeds were dry they were sown in the Jacobsen germinator.The control seeds were immersed in an aqueous suspension of the growth medium.There were four replications of each fungus (25 seeds/replication).The germination of the seeds and condition of the seedlings was checked 7 and 10 days after sowing.
Fresh peat was used as the growing medium in the greenhouse.An aqueous sus- was mixed into plastic boxes (50 X 25 X 9,5 cm), each containing 7 litres of peat.
In the control treatment a mixture of water and the growth medium was added to the peat.After being innoculated, the boxes were kept in the greenhouse for one week and the test seeds then sown in the boxes.The spring turnip rape varieties »Span» (0-variety), »WW-1644» (00-variety) and »Candle» (000-variety), and the spring rape variety »Oro» were used as the test plants.One row of seeds of each variety were sown in each box (30 seeds/row).The seedlings were grown until they reached the 3-4 leaf stage.The number of seedlings which deve- loped was counted weekly.The mean temperature throughout the experiment was + 20°C.At the end of the experiment the height of the seedlings from the root collar to the tip of the uppermost leaf was measured and the degree of infection determined ac- cording to the previously described criteria.

Statistical treatment
The number of seedlings suffering from pre-emergence damping-off was calculated in the pot experiment as the differences in emergence between t(ie control and test treat- ments.Seedlings killed by pre-emergence damping-off were placed in group 2 of the disease scale.
A value weighted on the basis of the culti- vation history of the field was given for the cultivation of oil seed crops.The more frequent and the closer to 1981 that oil seed crops had been grown in the field, the larger was the weighted value (Table 3).
The material was tested using the Stu- dent's t-test, analysis of variance and regres- sion analysis.The differences between the different treatments were analysed by means of LSD in cases where the F value obtained in analysis of variance was statistically signif-icant.The degree of statistical significance is denoted as follows: P < 0.05 = +, P < 0.01 = + + , and P < 0.001 = + + +.

Results
1.The symptoms of damping-off The initial symptoms of post-emergence damping-off are a partial darkening of the hypocotyl and radicle of the turnip rape or rape seedlings.As the disease progresses the whole of the hypocotyl becomes dark and shrinks to form a thread-like strand. (Fig. 1).The plants are either killed off by the post-emergence damping-off or recover to some extent as a result of secondary growth of the hypocotyl, but remain poorly devel- oped.Pre-emergence damping-off is only evident as a poor plant stand.

Incidence of damping-off
In the crop rotation experiment, spring turnip rape was grown as the first and second crop on the same experimental block in the longer crop rotation, and as the second and third crop in the shorter crop rotation during 1979-1982.The number of plants suffering from damping-off increased in both crop rotations as the number of times spring tur- nip rape was grown on the same site in- creased (Table 1).When turnip rape was grown for the second time on the same block the number of plants suffering from damping-off increased to about 20 °7o, and when it was cultivated for the third time the number of damping-off seedlings was already about 38 %.
Damping-off occurred on 56 of the 62 fields examined in 1981 and on 46 of the 50 fields in 1982.The mean number of seedlings affected by damping-off on the fields in each year was about 10 %.About 20 % of the fields in both years had a damping-off rate of 16-30 % of the seedlings, and 6 % of the fields had a damping-off rate of over 30 % (Fig. 2a).There were no regional differences as regards the incidence of damping-off.The mean number of seedlings suffering from damping-off in spring turnip rape fields was 9.5 %, and in spring rape fields 12 %.There were considerable differences in the inciden- ce of damping-off in the subsamples from the same fields.
It was also possible to estimate the amount of pre-emergence damping-off in the experiments in which seedlings were grown in the soil samples collected in 1981.The incidence Table 1.The occurrence of damping-off on spring turnip rape in the crop rotation experiment during 1979-1982.of damping-off was 18 % at the higher tem- perature, and 26 % at the lower tempera- ture.(Fig. 2b).The proportion of preemergence damping-off was considerably greater at the lower temperature than at the higher one (Table 2).
Information was collected in 1981 about the cultivation history of 38 fields.Spring turnip rape or spring rape had been grown at least once on most of the fields during the previous four years, which indicates that spring oil seed crops are very frequently grown on the same area (Table 3).On the ba- sis of the experiments involving the growing of seedlings at higher temperatures, the amount of damping-off increased as the weighted value for the cultivation of oil seed crops increases (Fig. 3).The incidence of damping-off varied between wide limits in the case where oilseed rape had been grown in the fields during the preceding four years.If oilseed rape had not been grown in the fields during the preceding four years, the incidence of damping-off was low.  1 1 = spring turnip rape or spring rape, 0) = other crop (mainly cereals).
2 The weighted value for the cultivation of oil seed rape was obtained by multiplying the value for the 1977 crop by 1, for the 1978 crop by 2, for the 1979 crop by 3 and the 1980 crop by 4, and then adding the results together.

Fungal isolations from plants suffering from damping-off
The fungus most frequently isolated from spring turnip rape and spring rape plants suf- fering from damping-off was Rhizoctonia solaniKiitin (Table 4).The fungus was iden- tified on the basis of the colour and morphological characteristics of the mycelia and sclerotia.There were no differences in the occurrence of Rhizoctonia solani in different years.

Pathogenicity tests
All the Rhizoctonia solani isolates obtain-  ed from the turnip rape and rape plants were pathogenic to the test plants (Fig. 4).They almost completely prevented the devel- opment of the seedlings on the peat substrate in the greenhouse (Table 5).In the case of the experiment with the Jacobsen germinator, three of the Rhizoctonia solani isolates were weaker pathogens than the other four which were isolated from turnip rape and rape.The Rhizoctonia solani isolate obtained from potato was not pathogenic in either of the tests.The Rhizoctonia solani isolate from Fig. 4. The pathogenicity of the fungal isolations to spring turnip rape and spring rape determined in a Jacobsen germinator and in peat substrate in the greenhouse.F values: Jacobsen germinator, fungi = 25,1***, Greenhouse, fungi = 100***, varieties = 3,5, fungi x varieties = 1,2.If there is the same letter at the top of two columns, then there is no significant difference (P = 0,05) as regards the degree of infection.
lettuce was pathogenic in the Jacobsen ger- minator but not in the peat substrate.On the other hand, the Rhizoctonia solani isolate from barley was pathogenic in the peat substrate but not in the Jacobsen germinator.
The interaction for pre-emergence damping-off between the fungi and the plant varieties in the pathogenicity test carried out in the peat substrate was statistically significant.Pre-emergence damping-off oc- curred most in Oro spring rape and least in the number variety WW-1644 (Table 5).Fusahum avenaceum caused the most pre- emergence damping-off to the Oro variety, and less damage to WW-1644 than to the oth- er varieties.The Rhizoctonia solani isolates obtained from potato and barley caused less pre-emergence damping-off to the WW-1244 variety than to the other varieties.However, there were no significant differences at the end of the test between the varieties of turnip rape and rape as regards the degree of infec- tion.
The seedlings grown in peat substrate infected with Rhizoctonia solani isolates were shorter (Table 6).All the fungi, apart from Fusahum sambucinum, clearly decreased the height growth of the seedlings in comparison to the controls.Although the Rhizoctonia solani isolates from potato and lettuce did not cause very much damping-off, they significantly reduced the height growth of the seedlings.

Discussion
Rhizoctonia solani Kiitin was found to be the most common and most pathogenic fungal pathogen causing damping-off on spring tur- nip rape and spring rape.The fungus is polyphagic and a number of strains with varying properties are known (Mordue  1974).In Finland, R. solani causes diseases on cereal and grass crops and on potato (Ylimäki 1967, Seppänen 1979, Mäkelä &   Parikka 1980).It has earlier been found to be a causal agent of damping-off in green- house cultivations (Linnasalmi 1952).R.
solani has also been found to be pathogenic to turnip rape and rape in Europe and in Canada (Raabe 1939, Berkenkamp& Vaart- nou 1974).After the seedling stage has been reached, R. solani has been frequently iso- lated from turnip rape and rape suffering from foot rot (Petrie 1974, Kruger 1979).
All the tested R. solani isolates from spring turnip rape and spring rape were ex- ceedingly pathogenic.The difference between the pathogenicity of these isolates in the Jacobsen germinator may be due to the nutrient requirements of the isolates.An ex- ternal source of nitrogen is an important fac- tor in infection by R. solani (Weinhold et   al. 1972).In the case of the peat substrate, which is rich in nitrogen, there were no differences between the isolates obtained from spring turnip rape and spring rape, and the isolate obtained from barley was also pathogenic.Behaviour of the isolate obtained from lettuce was, on the other hand, quite the opposite as it was pathogenic only in the Jacobsen germination.The isolate from potato was not pathogenic to turnip rape or rape.This supports the generally held belief concerning the specialised nature of R. sola- ni isolates from potato (Parmeter et al.  1969).
As far as the other tested fungi were con- cerned, only Fusahum avenaceum Sacc.was clearly pathogenic to turnip rape and rape.However, owing to the fact that it oc- curred in very small numbers only, its importance as a causal agent of damping-off was small.As it appears, according to Tah-  vonen (1979) and the results of this study concerning the isolations made on plants af- LSD (P = 0,05) fungi = 3,4 %, varieties = 2,0 % and variety with the same fungus = 8,2 %.Table 6.Effect of the fungal isolates on the height growth of spring turnip rape and spring rape in the pathogeni- city tests carried out on the peat substrate.

Fungus
Seedling height (cm)    1 If two mean values are followed by the same letter, then the means do not differ significantly (P = 0,05) from each other.
fected by damping-off, that the importance of seed-borne fungi is of only slight importance in Finland, thiram dusting which is widely used here is of dubious usefulness.
No information was obtained about preemergence damping-off when collecting the plant samples from the experimental plots and the fields used in commercial farming.
In Linnasalmi's (1952) experiments, for tion experiment and the field observations, instance, R. solani caused a considerable amount of pre-emergence damping-off.The proportion of pre-emergence damping-off was considerably large in the case of seed- lings grown in soil taken from fields, espe- cially at a low temperature.The low tempe- rature used in the experiments is quite equi- valent to the mean soil temperature after sowing dates in Finland.This indicates that a plant stand of spring oilseed rape may be re- duced as a result of pre-emergence dampingoff.The large variance in the subsamples also indicates that the pathogen is present in the soil in patches.The patchy occurrence of the disease caused by R. solani is due to the growth pattern of the fungus in the soil (Baker et al. 1967).In addition to reducing the plant stand, the initial development of the seedlings can also be slowed down.This was reflected in the pathogenicity tests as slower growth of the seedlings.R. solani has been found to slow down the initial develop- ment of soy beans, although there were no clear symptoms of the disease (Grau & Mar- tinson 1979).The sparse stands of turnip rape and rape, and their slow initial development, weakens their ability to compete with weed plants (Köylijärvi and Tulisalo 1982).
According to the results of the crop rota- the incidence of damping-off clearly in- creased as the number of times spring turnip rape and spring rape were grown on the same site increased.It was possible to explain 27 % of the variation in the incidence of damping-off in the fields by means of the weighted value for oil seed crops.In the crop rotation experiment, the incidence of damping-off increased in both rotations, although spring turnip rape was grown on the same area only every fourth year in the longer rotation cycle.Furthermore, the R. solani isolate obtained from barley was pathogenic to spring turnip rape and to spring rape.These observations indicate a need for further studies into the behaviour of R. solani in cereal oilseed rape rotations.In addition, the role of R. solani as a causal agent of foot rot on spring oilseed rape in Finland should also be studied, as well as the effect of Rhizoclonia diseases on the yield from spring oilseed rape.mipoltteen suuruus.Matalassa lämpötilassa kuoli maanalaiseen taimipoltteeseen keskimäärin 14.9 % taimista, mikä oli merkittävästi enemmän kuin korkeassa lämpötilassa maanalaiseen taimipoltteeseen kuolleiden taimien luku (4.8 %).

Fig
Fig. I. Damping-off caused by Rhizoctonia solani on turnip rape, a) on germlings, b) large damaged plants.

Fig. 2 .
Fig. 2. The abundance of damping-off on spring tur- nip rape and spring rape in 1981 and 1982.A) on the basis of seedling samples taken from the fields, B) in seedling growing experiments car- ried out at temperatures of 25 °C and 11°C in soil samples collectedin 1981.

Fig. 3 .
Fig. 3. Dependence of the incidence of damping-off on the cultivation of oil seed crops.The damping-off observations are from seedling growing experiments using soil samples at 25°C.The figures are for both pre-emergence and post-emergence damping-off.y = 5.15 e 0 24 », r = 0.52 + + +

Table 2 .
Effect of a high and a low growing tempera- ture on the amount of damping-off in spring turnip rape seedlings.Mean of 30 soil sam-

Table 3 .
Distribution of the number of times oil seed crops were grown and the weighted value for the cultivation of oilseed rape in the sample fields in 1981.

Table 4 .
The fungi most frequently isolated from turnip rape and rape suffering from damping-off.
% of the isolations

Table 5 .
Pre-emergence damping-off on turnip rape and rape in the pathogenicity tests carried out in the peat substrate.