RELATIONSHIPS BETWEEN SEED DEVELOPMENT STAGE, GERMINATION, OCCURRENCE AND LOCATION OF FUNGI IN OILSEED RAPE (BRASSICA NAPUS SSP. OLEIFERA L.) SEEDS AND THE PRESENCE OF ALTERNARIA AND CLADOSPORIUM SPP. SPORES IN THE AIR

Dorota Szopińska¹, Krystyna Tylkowska¹, Alicja Stach^2
1 Department of Horticultural Seed Science and Technology, Agricultural University of Poznan, Poland
² Laboratory of Aeropalynology, Adam Mickiewicz University, Poznan, Poland

ABSTRACT

The study performed in the years 2002-2004 determined seed germination and the occurrence of fungi on and in the developing seeds of oilseed rape. What was also examined was the correlation between the occurrence of Alternaria and Cladosporium spp. spores in the air and seed infestation with the fungi. The highest values of seed germination were observed in 2003. The composition of fungi varied depending on the year and the stage of seed development. Irrespective of the year and the position on a plant, Alternaria and Cladosporium spp. were the dominating fungi detected on the oilseed rape seeds. The majority of fungi contaminated the seed surface. Cladosporium spp. occurred at high percentages from the early stages of seed development, whereas the highest percentages of Alternaria spp. were detected at the end of seed maturation. The number of fungi infecting the seed coats and the embryos increased during the seed development. Towards the end of maturation, more fungi were detected in the seed coat and the embryo of the seeds from the secondary branches as compared with those obtained from the main racemes. The spores of Cladosporium and Alternaria spp. were present in the air throughout the whole observation period. The number of the Alternaria spp. spores in the air increased with time, reaching the highest values at harvest. Cladosporium spp. spores occurred in the air at high concentrations from the very beginning of the observation period. The highest concentration of Cladosporium spp. spores in the air was recorded in 2004. The correlation coefficients between the number of Alternaria and Cladosporium spp. airborne spores and the percentages of the seeds infested with the fungi were not statistically significant.

Key words: oilseed rape, seed development stage, seed germination, location of fungi, airborne spores.

INTRODUCTION

Oilseed rape (Brassica napus L. ssp. oleifera Metzg.) seeds are the main source of oil for the food industry in Poland. Furthermore, an increasing bio-fuel production offers new opportunities for rapeseed producers. This means that the cultivation land for oilseed rape could be extended. Frencel et al. [8] suggested that there was a clear relation between the severity of fungal diseases and the intensity of crop cultivation, i.e. between the size of the land cultivated for oilseed rape and the frequency of crop rotation.

The fungi detected in oilseed rape seeds included both pathogenic and saprotrophic species. Alternaria brassicae and Leptosphaeria maculans (anamorph Phoma lingam) belong to the most important pathogens. Moreover, the economic significance of Sclerotinia sclerotiorum and Botrytis cinerea has been proved [8]. Seed yield losses due to A. brassicae may be greater than 50% and even the occurrence of a moderate disease may cause losses of 20%. Additionally, an infection by the fungus may reduce the oil content in seeds by up to 35% [3,12]. Apart from A. brassicae, two other species, Alternaria alternata and A. brassicicola, are responsible for Alternaria blight in oilseed rape. All these species produce almost identical symptoms, which are visible as spots on leaves, shoots and siliques, though varying in intensity. While A. alternata colonizes equally all parts of plants, A. brassicicola infects mostly siliques and stems, whereas A. brassicae infects mainly leaves and siliques [16]. The relative harmfulness of Alternaria blight (Alternaria spp.) and grey mould (B. cinerea) varied in different years depending on the local conditions and the weather [8].

The seed coat is the common site of an infection for most seed-transmitted Mitosporic fungi (formerly Fungi Imperfecti). Such infections, established as dormant mycelium, have been demonstrated for A. brassicae, B. cinerea and P. lingam. The latter has frequently been found in the embryo of cabbage seeds [20]. A. brassicae may be carried onto the surface of seeds and as internal infections [12,18]. According to Maude and Humpherson-Jones [18] high levels of an internal infection indicate that the fungus was not just a random contaminant, but that its presence resulted from a direct attack of siliques.

The concentration of spores in the air is, in general, closely correlated with the crop production in the area [19]. Fungal spores are probably a significant factor causing different types of allergy when the spores are inhaled and deposited on the sensitive mucosa [9]. Cladosporium spp. belongs to the most common spore-type in the air in many parts of the world. Most reports concern C. herbarum, the amount of which in the air may sometimes reach as much as several thousand spores per cubic meter. Other common spores in the outdoor air include conidia of Alternaria, Botrytis and Epicoccum spp. [13].

Monitoring the changes in the occurrence of fungi in the developing seeds of oilseed rape was the main aim of the study. Moreover, the concentration of Alternaria and Cladosporium spp. spores in the air and seed infestation with the fungi was investigated.

MATERIAL AND METHODS

The investigations were conducted in the years 2002 to 2004 on the Brassica napus ssp. oleifera cv. Lirajet, a winter variety cultivar of oilseed rape, grown in the fields located in Baranowo (N = 52°12’54.7’’, E = 16°55’12.5’’) in the 2002, and in Cerekwica (N = 52°31’15.16’’, E = 16°41’30’’) in 2003 and 2004 – in the Wielkopolska region, Poland.

Germination and mycological analyses were performed on the seeds collected from the main racemes and the secondary branches at one week intervals, starting three weeks after the anthesis until the harvest. Daily average fungal spore concentrations were measured with a Hirst volumetric spore trap (Burkard, UK), which was placed on a roof in the urban area of Poznań, Poland, at the height of 33 m (N = 52°24’22.5’’, E = 16°55’24.5’’).

Seed germination capacity
Seeds from both the main raceme and the secondary branches were analyzed, 300 of which had been pre-treated with sodium hypochlorite (NaOCl) and another 300 had not. The surface of the seeds was disinfected with a 1% aqueous solution of NaOCl for 10 min, followed by rinsing it three times in sterile distilled water and drying with sterile blotting paper. 50 seeds from each category were placed in Petri dishes containing six layers of moistened blotters and were incubated in darkness at 20°C. The action was repeated six times. In accordance with the ISTA guidelines [14], the percentage of normal seedlings (germination capacity at the second count) was determined after ten days.

Mycological analysis
200 seeds (20 seeds per a 9 cm diameter Petri dish) from the main raceme and 200 seeds from the secondary branches were disinfected and tested using the blotter method. The seeds were disinfected with a 1% aqueous solution of NaOCl for 10 min. Then they were rinsed three times with sterile distilled water and dried with sterile blotting paper. The seeds were incubated for 24 hours at 20°C in darkness. After that, they were transferred to -20°C for 20 hours and incubated at 20°C at alternating cycles of 12 hours of near ultraviolet (NUV) light and 12 hours of darkness for eight days. The fungi were identified on the basis of their growth and sporulation using a stereoscopic and compound microscopes. The total number of seeds infested with fungi and the number of seeds infested with particular fungi were determined. The same analysis was performed for non-disinfected seeds. The conclusion about the location of fungi in the seeds was drawn on the basis of the differences in their occurrence in disinfected and non-disinfected seeds.

Component plating
The seeds were disinfected with a 1% aqueous solution of NaOCl for 10 min; and then they were rinsed three times with sterile distilled water. 100 seeds from the main raceme and 100 seeds from the secondary branches were tested. Each seed was dissected aseptically into the embryo and the seed coat under a stereoscopic microscope. The components were placed on a potato dextrose agar medium (PDA, Scharlau Chemie, Spain) in a 9 cm diameter Petri dish, two sectioned seeds per dish. 100 ppm of streptomycin was added to the medium to prevent the development of bacteria. Petri dishes were placed at 20°C at alternating cycles of 12 hours of NUV light and 12 hours of darkness for 10 days. The fungi which had grown around particular parts of the seeds were identified on the basis of their growth and sporulation visible under a stereoscopic and compound microscopes.

Fungal spore concentration
The measurements were taken with a Hirst volumetric trap (Burkard, UK) [10]. This method allows to determine the daily average concentration of fungal spores in the air [1,7,17]. The trap sucked 10 dm³ of air per minute. The inlet of the trap was 2 mm x 14 mm in size. Bioaerosols were deposited on a Melinex tape covered with a sticky medium. The tape was placed on a drum rotating with a constant speed of 2 mm per hour. The tape was replaced once a week, and then it was cut into segments corresponding to 24 hours and microscopic slides were prepared.

The average daily concentrations were obtained by counting the spores every hour along two horizontal transects and by applying an appropriate conversion factor (F) which was calculated as follows:

where:
a – total area of glass (14 × 48 mm = 672 mm²),
b – analysed area (0.60 mm × 48 mm × 2 = 57.6 mm²),
c – volume of analysed air (10 dm³·min^-1 = 14.4 m³·24 h^-1).

Meteorological data
Daily meteorological data, i.e. the measures of the meanmean air temperature, the relative humidity and the total amount of precipitation, were obtained from the Poznań Institute of Meteorology.

Statistical analysis
The results obtained were prepared using the variance analysis method and the significance of the differences between the mean values was evaluated by Duncan’s multiple range tests. Before the analysis the percentage values were transformed in accordance with the Bliss function. The relationships between the concentration of Alternaria and Cladosporium spp. spores in the air and seed infestation with these fungi were determined using the analysis of linear regression. Also, the correlation coefficients were calculated.

RESULTS

In many cases, disinfection improved the germination capacity of seeds. This applies both to the seeds from the main raceme and to those from the secondary branches, especially at the end of the evaluation. In 2003 the highest values of the parameter were observed, especially for disinfected seeds. In general, in the years 2003 and 2004 the seeds from the main raceme germinated better than the seeds from the secondary branches. The situation was opposite in 2002, particularly at the end of vegetation (Table 1).

The total number of the seeds infested with fungi varied during seed development (Table 2). It was observed that each year, especially for the seeds from the secondary branches, the number of infested seeds was much higher at the end of vegetation compared to their number on the first date of the evaluation. The composition of fungi varied depending on the year and stage of seed development. The following fungi were identified on the tested seeds: Alternaria alternata (Fr.) Keissler, Alternaria brassicae (Berk.) Sacc, Botrytis cinerea Pers. ex Pers., Cladosporium spp., Epicoccum purpurascens Ehrenb. ex Schlecht., Fusarium spp., Gonatobotrys simplex Corda, Mucor spp., Penicillium spp., Phoma spp., Rhizopus sp., Stemphylium botryosum Wallr., Trichoderma spp., Ulocladium consortiale (Thüm) E. Simmons and Verticillium spp. A special attention was paid to those of the greatest importance for oilseed rape and to those occurring in high percentages (Tables 3, 4, 5 and 6).

Irrespective of the year and the position on a plant, Cladosporium and Alternaria spp. were the dominating fungi detected in the seeds. The number of seeds infested with A. alternata increased gradually from the beginning to the end of seed development (Tables 3 and 4). However, the occurrence of Cladosporium spp. in and on the seeds was not clearly related to the stage of seed development (Tables 5 and 6). With a few exceptions, disinfection significantly decreased the number of seeds infested with the fungi, especially at the end of the evaluation. A. brassicae occurred in the years 2002 and 2004, during the last weeks of vegetation and, in general, there were no differences in the infestation with the fungus for non-disinfected and disinfected seeds. B cinerea was observed mainly in 2002 and, initially, was present only on the seed surface. The number of infested seeds decreased gradually during the observation period but, at the same time, the level of internal infection increased (Tables 3 and 4). Phoma spp. occurred occasionally, regardless of seed treatment and their position on a plant.

The differences in the occurrence of fungi on disinfected and non-disinfected seeds indicated that the majority of fungi contaminated the seed surface. However, component plating, showed that the number of fungi infecting the seed coat and the embryo increased with seed development. Cladosporium spp. occurred in the seeds at the early stage of seed development, whereas Alternaria spp. was detected particularly at the end of vegetation. Towards the end of seed development, more fungi were detected in the seed coat and the embryo of the seeds from the secondary branches than in the seeds from the main racemes. Component plating revealed that A. brassicae was mainly located in the seed coat (Table 7).

The spores of Cladosporium and Alternaria spp. were present in the air throughout the whole observation period. Both the percentage of seeds infested with Alternaria spp. and the number of spores of the fungi in the air increased with time, reaching the highest values during the final seed analyses. Then the concentration of Alternaria spp. spores exceeded 100 spores per 1 m^-3 each year and in the years 2002 and 2004 it was even higher than 250 spores per cubic meter (Fig. 1). The highest concentration of Cladosporium spp. spores in the air was recorded in 2004. The value did not fall below 3,500 spores per 1 m^-3 during the observation period and reached 18,364 spores per m^-3 at the end of it. The concentration of Cladosporium spp. spores was much lower in the previous years and ranged from 528 to 6,241 spores per 1 m^-3, and from 430 to 9,236 spores per 1 m^-3 for 2002 and 2003, respectively. There was no clear relationship between the percentage of seeds infested with Cladosporium spp. and the number of spores present in the air (Fig. 2).

Seasonal weather patterns varied substantially in different years (Table 8). Year 2002 was characterized by the highest relative humidity, combined with the lowest air temperature at the beginning of the observation period and the lowest total amount of precipitation. The next year the highest mean air temperature and the lowest relative humidity were reported. However, in 2004 the total amount of precipitation was higher and the mean air temperature was lower than in the years 2002 and 2003. Neither the air temperature nor the relative humidity or precipitation was significantly related to Alternaria and Cladosporium spp. spore concentration in the air (Table 8, Fig. 1 and 2).

DISCUSSION

The highest values of seed germination capacity were observed in 2003. That was also the only season when A. brassicae was not detected in the tested seeds. The season was characterized by a higher mean temperature and a lower relative air humidity, which favoured faster seed ripening; and that, in turn, increased the germination capacity and could be the reason for the absence of A. brassicae. According to Maude and Humpherson-Jones [18] brassica seeds infested with A. brassicae and A. brassicicola are often shrivelled and have a low viability. Norton and Harris [21] observed that the development of oilseed rape seed appeared to be complete two weeks before harvest and in the remaining period only dehydration occurred. In the present experiment, the results of the germination test for the last two dates of evaluation were comparable, especially for the disinfected seeds. This may suggest that two weeks before harvest the process of seed development was completed and the differences in germination capacity were caused by an increasing seed infestation with fungi.

In 2002, higher relative air humidity than in the years 2003 and 2004, combined with a low temperature at the beginning of the observation time, could favour a higher seed infection with Botrytis cinerea. Only in this year the pathogen was detected both in the seed coat and in the embryo, regardless of the seed position on the plant. Moreover, as early as at the beginning of the observation period the pathogen was observed in the seed coat of the seeds from the main raceme.

Alternaria alternata and Cladosporium spp. were the most frequent fungi recovered from rape seeds, regardless of the season. A common occurrence of these species on oilseed rape seeds was indicated by several authors [4,6,23,26].

A high level of seed infestation with Cladosporium spp., occurring from the beginning until the end of seed development, could have resulted from the ability of the genus to grow under drier conditions compared to many other fungi, including Botrytis, and therefore to its ability to colonize dead tissues more readily [20]. The highest seed infestation with Alternaria spp. observed at the end of vegetation was probably caused by a secondary infection with the fungi and could also be caused by an increasing susceptibility of plants at this stage of development. According to Neergaard [20], A. brassicae may penetrate the silique and afterwards move on directly inside the maturing seeds thus infecting any part of the seed coat. The fungus causes black spots on the stems and the siliques of oilseed rape. A consequence of an infection may be a severe damage and yield losses may result from flower bud abortion, premature ripening, siliques shattering and seed shrivelling [3]. The two latter symptoms were observed in the experimental fields in 2004 when the level of an infection was much higher than in 2002. A. brassicae was found both in disinfected and non-disinfected seeds at the end of seed development, although component plating showed the presence of the fungus in the seed coat of oilseed rape only in 2004. Shrestha et al. [24] found A. brassicae predominantly in the seed coat and rarely in the embryo of rapeseed (Brassica campestris L. var. toria) and mustard (Brassica juncea).

Neergaard [20] pointed out that Phoma lingam was frequently found in the embryo of cabbage where the hyphae were usually limited to the edges of peripheral layers of cotyledons, but occasionally the radicle was also infected. In the present experiment Phoma spp. was found both in the seed coat and in the embryo of the examined seeds. Towards the end of vegetation, more fungi were detected in the seed coat and the embryo of the seeds from the secondary branches than in those from the main racemes. Presumably, less developed seed coat tissue of the first could not act as a mechanical barrier limiting a fungal penetration [20,25].

The spores present in the air may be brought to the earth by sedimentation attracted by the gravity, may be carried by air currents and deposited or may fall down with rain. Very often these processes are combined. [13]. The Wielkopolska region is characterized by strong western winds and a rather dry climate with a limited amount of rainfall. Such conditions favour the release of spores and their dispersion over a large area. The inoculum of Alternaria species is mainly distributed by airborne dispersal, although splash dispersal is occasionally possible. Wind, combined with low humidity, is the main factor in releasing spores from plants [22].

Fungal spores from infested plants may be a significant source of human allergies [2,5,15,27]. For two of the most common genera of allergenic fungi, the threshold concentrations causing allergic reactions are estimated at 100 and 3,000 spores per 1 m^-3 of the air for Alternaria and Cladosporium, respectively [9]. In our experiment, these thresholds were exceeded considerably throughout the observation period, which was noticed especially for Cladosporium spp. When working on cotton, Mitakakis et al. [19] found that crop maturation affected the Alternaria spore content in the air. The concentration of spores above crops was higher during maturation and harvest than at the time when the crops were immature. This seemed to be in agreement with our data, which showed a higher concentration of Alternaria spores in the air at the end of the vegetation period. According to Rotem [22], all Alternaria species are highly resistant to adverse weather conditions. They develop in a wide range of temperatures using the locally available sources of moisture, and they produce a relatively small number of spores, mainly at the end of the season. This situation may be hazardous both for plants and humans; on the one hand, the spores in the air could be the source of an infection for cultivated plants and, on the other hand, they may cause respiratory diseases in the population of farm workers and the inhabitants of the neighbouring towns. According to Humpherson-Jones [11] A. brassicae airborne spores are the main source of inoculum and they may be dispersed over considerable distances. A. brassicae occurs commonly on cruciferous weeds and forage brassicas and the isolates of the fungi from such species are virulent on ware brassicas. The experiment of Mitakakis et al. [19] showed that the overall spore concentrations above towns in rural areas were related to the concentrations above the crops mentioned earlier. Does the level of plant infection, and in consequence seed infestation with fungi, depend on the spore concentration in the air or is the high spore concentration in the air caused by severe plant infection? It seems to be obvious that this relationship is mutual.

CONCLUSIONS

1. The highest values of seed germination capacity were observed in 2003 which was characterized by higher mean temperature and lower mean relative humidity of the air than in the years 2002 and 2004.

2. The composition of fungi varied depending on the year and the stage of seed development. Irrespective of the year and the position on a plant, Alternaria and Cladosporium spp. were the dominating fungi detected on the oilseed rape seeds. Cladosporium spp. occurred at high percentages from the early stages of seed development, whereas the highest percentages of Alternaria spp. were detected at the end of vegetation.

3. The majority of fungi contaminated the seed surface. The number of fungi infecting seed coats and embryos increased during seed development. At the end of vegetation, more fungi were detected on the seed coat and the embryo of the seeds from the secondary branches than on those from the main racemes.

4. The spores of Cladosporium and Alternaria spp. were present in the air throughout the whole observation period. However, the relationships between the percentage of seeds infested with Alternaria and Cladosporium spp. and the number of their spores present in the atmosphere were not statistically significant.

ACKNOWLEDGEMENTS

We would like to thank Dr Małgorzata Jędryczka and the Institute of Plant Genetics in Poznań for making available for us their experimental fields.

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Accepted for print: 23.10.2007

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