ABSTRACT

Stem rot, caused by Sclerotinia sclerotiorum, is an important disease of oilseeds and often causes severe losses. The pathogen is dispersed by airborne ascospores, which in oilseed rape do not usually infect plants directly but via petals deposited on leaves or stems. However, little is known of the conditions under which successful infection takes place. This paper reports the results of controlled environment studies of the infection processes of S. sclerotiorum on oilseed rape plants. Temperatures between 20 and 25°C and relative humidities (RH) >80% were required for optimal plant infection. At 20°C and c.100% RH lesion size, measured six days after inoculation, increased with inoculum load up to about 80 ascospores per leaf, larger ascospore loads did not increase lesion size. Petal age appeared to affect the efficiency of infection. Lesions were significantly larger and less variable for “old” petals compared to “young” petals. Infected-petals, which were dried prior to leaf-inoculation produced much smaller lesions than petals which were placed on leaves in water drops. About two days of high humidity was needed for lesion initiation in some oilseed varieties. The results suggest that factors such as temperature, humidity, inoculum load, and water availability may play important roles in epidemic development in the field.

INTRODUCTION

Sclerotinia stem rot caused by the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary is one of the most damaging diseases of oilseed rape (Brassica napus L ssp. oleifera). The fungus over-winters in the soil as sclerotia and, under favorable environmental conditions, these can germinate carpogenically to produce ascospore-bearing apothecia. Ascospores are released under conditions of changing humidity but require a substrate on which to germinate before they can infect their host. In oilseed rape this substrate is usually petals. Plant infection takes place when ascospores deposited on petals germinate and the petals fall and adhere to the plant. However, little is known of the conditions required for either ascospore infection of petals or subsequent plant infection. This paper reports the results of controlled environment studies on environmental factors affecting oilseed rape infection by ascospore infected petals.

MATERIALS AND METHODS

Experiments were done in controlled environment (CE) chambers using 40-45 day old greenhouse grown (20±2 °C and 16 hours photoperiod) oilseed rape plants. Inoculated plants were placed in a “humidity chamber” within the CE chamber. The humidity chamber consisted of a metal frame covered in polythene sheeting and allowed humidities close to saturation to be maintained.

Plants were inoculated by placing five petals infected with ascospores on a single leaf. The petals were infected with ascospores as follows. Apothecia of S. sclerotiorum were produced from sclerotia (isolate collected from winter oilseed rape at IACR-Rothamsted) using the method described by Sansford and Coley-Smith (1992). Ascospores were collected on cellulose filters mounted in 37mm filter holders (Millipore Ltd., Watford, U.K) using a vacuum pump. The filters were stored in a freezer if they were not required immediately. Ascospore suspensions were made by shaking a filter in 5ml of sterilized distilled water. After removing the filter the ascospore concentration in the suspension was adjusted to c. 2 x 10⁴ ascospore ml^-1. Petals were infected by adding eight petals per ml to the ascospore suspension, and incubating at 20°C for 3 hours.

Disease severity was recorded as a lesion index (LI) given by the sum of lesion length (cm) and lesion width (cm). LI was used as it was affected less by lesion shape than single length or width measurements (long thin lesions and short wide lesions were weighted equally with this index).

Environmental factors.

All experiments on the effects of temperature, relative humidity and leaf wetness were done with spring type cultivar Rebel. The experiments on humidity duration were done with Chinese cultivars Y16, PAUC61, 821 & 681. Five replicates were used per treatment in each experiment.

Temperature. Inoculated plants were placed in the humidity chamber at different temperatures (15, 20, 25 & 30 °C) at c.100% relative humidity (RH). Observations were made daily and the time of lesion initiation and time taken for LI to reach 20 were recorded.

Relative humidity. Inoculated plants were placed in the humidity chamber at different RH values (50, 80 & 100 %) and a temperature of 20±2 °C. LI were measured after six days.

Humidity period. Inoculated plants were placed in the humidity chamber at c.100 % RH, and 20±2 °C. After different periods of time (16, 24, 48, 72, 96 or 144 h) the plants were moved to a chamber at 60 % RH and 20±2 °C. Lesions were assessed after six days.

Leaf-wetness. Ascospore inoculated petals were dried by placing them on filter paper. Plants were inoculated either with “dry” petals placed directly on leaves or “wet” petals placed in a droplet of water. The plants were kept at c.100% RH and 20±2 °C and lesions assessed after six days. Tests were done with petals that had or had not been incubated for 3h before placing them on leaves.

Inoculum density and petal characteristics.

All experiments were done at 20±2 °C and c. 100% RH using plants of cultivar Rebel. Lesions were assessed after six days and five plants were used in each treatment in each experiment.

Ascospore ‘load’. Petals were incubated in ascospore suspension of different concentration (0, 100, 500, 1000, 5000, 10000 & 20000 ascospores ml^-1) for 3 hours. Plants were leaf-inoculated with petals from each concentration, placed in three arrangements (over-lapping, touching or equally spaced).

Petal number. Plants were inoculated with 1, 2, 3, 4, 5, 7 or 10 ascospore-infected petals placed overlapping on the leaf. Lesions were assessed 4 and 7 days after inoculation.

Petal age & condition. Plants were inoculated with petals from newly opened buds (young petals) or from pollinated flowers (old petals) or with naturally fallen petals. Inoculations were also done with frozen and freeze-dried old and young petals.

Inoculation site. Plants were inoculated on stem, node, and leaves. LI was measured for leaf inoculated plants and lesion presence was recorded for stem- and node-inoculated plants.

RESULTS AND DISCUSSION

Environmental factors.

Temperature. Lesions were produced at all temperatures tested. Lesion initiation time decreased with increase in temperature up to 25 °C but was longer at 30 °C than 25 °C (Table 1). Similarly, the time to produce LI values of 20 decreased with temperature up to 25 °C but increased at 30 °C. Relatively large lesions were present over a wide temperature range (15-30°C), suggesting that in many oilseed growing regions temperature may not limit infections as long as there is high humidity.

Table 1. The effect of temperature on lesion development on plants (cv. Rebel) inoculated with S. sclerotiorum. Relative humidity was maintained at c.100%.

Temperature (°C)	Lesion initiation (days)	LI=20 a (days)
15	4-5	8-9
20	2-3	6-7
25	2-3	5-6
30	3-4	6-7

^a Lesion Index (sum of lesion length (cm) and width (cm)) equal to 20

Relative humidity. Lesions only developed when relative humidity was close to 100% RH (mean LI=18.3, sd=0.76, n=5). At 50 % and 80 % RH the petals dried after 24 hours and no lesions developed. Humidities greater than 80% appear to be essential to allow ascospore infected petals to infect leaves, possibly by preventing the petals drying before leaf infection takes place. Little is known of the effect of re-wetting petals on infections.

Humidity period. Except for one replicate of line 821, no lesions developed on plants kept at high humidities for less than 48h. After 48h, LI tended to increase with time under high humidity for all lines tested. However, LI values differed between lines: e.g. mean LI after 96h of c. 100% RH were 12.1, 10.8, 10.0 and 6.6 for lines PAUC61, Y16, 681 and 821 respectively. This suggests that periods of high humidity of up to 48h may be required to initiate infections.

Leaf-wetness. Lesions developed on all plants regardless of whether the petals were “wet” or ”dry”, suggesting that liquid water may not be essential for infection provided that humidities are high. However, lesions were significantly larger for “wet” petals than “dry” petals: mean LI values for incubated petals were 18.4 and 8.0 for “wet” and “dry”; and 14.7 and 5.5 “wet” and “dry” non-incubated petals. Thus, leaf wetness may aid lesion development. The reasons are not clear, but the presence of water may allow greater utilization of nutrients; or it may provide a medium within which the mycelium can grow from the petal to the leaf.

Figure 1. The effects on leaf infection of ascospore load and petal position

Inoculum density and petal characteristics.

Ascospore ‘load’. Lesion size increased with increasing ascospore concentration up to about 500 ascospores ml^-1 (Figure 1), but further increases did not increase lesion size. Petal position did not affect lesion size at concentrations above 500 ascospores ml^-1, however, at concentrations of 100 ascospores ml^-1 overlapping petals produced lesions two times larger than those produced by ‘touching’ or ‘spaced petals’. This may be due to greater localized leaf-wetness and ascospore concentration. A concentration of 500 ascospores ml^-1 corresponded to about 80 ascospore per leaf.

Figure 2. Petal number effects on leaf infection.

Petal number. Lesions developed in all treatments (Figure 2). However, after 7 days, lesion size increased with petal number up to four petals, further increases in petal number produced little difference in lesion size.

Table 2. The effect of petal age and condition on lesion development.

Petal age / condition	Lesion Index range	Mean Lesion Index	S.D a	S.E
Old / fresh	18 – 20.5	19.6	1.29	0.58
Young / fresh	0.4 – 15	6.7	5.28	2.36
Old / frozen	17 – 22	19.8	2.28	1.02
Young / frozen	0 – 20	11.0	7.97	3.56
Old & Young / frozen	0.8 – 20	7.3	7.98	3.57
Old / freeze dried	15.5 – 20	17.4	1.71	0.76
Young / freeze dried	6 – 20	14.0	5.61	2.51
Old fallen / dried	17 – 20	18.6	1.19	0.53

^a Standard deviation, n=5.

Petal age & condition. Old petals produced relatively large lesions of consistent size, whereas young petals produced smaller more variable lesions (Table 2). The condition of old petals, prior to inoculation, had little effect on lesion size, however, lesions produced by frozen and freeze dried young petals were almost double the size of those produced by fresh young petals.

Inoculation site. Only one lesion (LI=0.2) developed after stem inoculation (5 plants). All node-inoculated plants became infected. Leaf-inoculated plants produced the largest and most consistent lesions (LI=19.3, s.d= 1.64, n=5).

CONCLUSIONS

An understanding of the interactions between environment factors and infection processes will improve the management of stem rot through the development of better forecasting systems. The results presented here can be of use in developing of new, more realistic, screening methods based on petal infection.

ACKNOWLEDGMENTS

This research was supported by a grant from the European Union, contract number ERBIC18CT970173. IACR-Rothamsted is supported by a grant in air from the UK Biotechnology and Biological Sciences Research Council.

REFERENCES

1. Sansford, C.E. & Coley-Smith, J.R. (1992). Production and germination of sclerotia of Sclerotinia sclerotiorum. Plant Pathology 41, 154-156.