Bekæmpelsesmiddelforskning fra Miljøstyrelsen, 96 – Bekæmpelse af sygdomme i frøbede af Nordmannsgran ved hjælp af biologiske og kulturtekniske metoder i skovplanteskoler

Bekæmpelse af sygdomme i frøbede af Nordmannsgran ved hjælp af biologiske og kulturtekniske metoder i skovplanteskoler

Summary

Damping-off during nursery production of conifer seedlings is a serious problem in Denmark. Soil-borne fungal infections can result in mortality of pre-emergence seedlings as well as post-emergence symptoms. Seed-borne fungi such as Fusarium spp., which can proliferate during stratification of forest seeds, can also add to the overall problems in the field. Fungal diseases can be partly controlled using fungicide seed treatments and especially by use of soil fumigants. However, particularly from an environmental point of view, alternatives to the very intensive soil fumigation are needed. Caucasian fir (Abies nordmanniana) plants are produced on a large scale as Christmas trees, and seedling production is the first phase of this production involves very high levels of pesticide use.

The objectives of this work were to: (i) identify the major pathogens involved in seedling diseases of Caucasian fir in the nursery, (ii) test the impact of different biological (antagonistic) seed treatment measures on seedling emergence and survival in the field and (iii) optimize biocontrol methods in combination with the available seed technology. Four different antagonists were included: three commercial products: 1)TRI 003 and 2) Supresivit, both based on the fungus Trichoderma harzianum and 3) FZB Biotechnik GmbH9, based on the bacterium Bacillus subtilis. Additionally 4), the near commercial fungal isolate Clonostachys rosea isolat IK726 was included as it has shown good effect in other cultures and stimulation of seedling emergence when coated on seeds in different wet-seed technologies such as seed priming of carrots. The field experiments were carried out at Fosdal Nursery under the Danish Forest and Nature Agency, where pesticide-free production was being implemented in 2002.

By applying baiting technique and pathogenicity testing of isolates on soil samples from the nursery, it was demonstrated that damping off was caused by soil-borne Pythium ultimum var ultimum and other Pythium spp., while post-emergence diseases such as top decay were caused by Fusarium oxysporum and Fusarium redolens. The most important seed borne fungi was Fusarium oxyporum. Also Papulaspora immersa, although not previously reported to be pathogenic, caused diseased seedlings in an inert growth substrate (vermiculite). Seed lots of varying vigour and dormancy were stratified for three and six weeks respectively and coated with C. rosea IK726 before and after stratification. Stratification was carried out at 3°C and 33% seed moisture content. In general, treatment with C. rosea IK726 after imbibition at the beginning of the stratification period, resulted in improved germination. Moreover, the treatment resulted in a significantly higher number of healthy seedlings in the inert medium, vermiculite.

Investigation into survival of C. rosea IK726 in different formulations showed that coating with either peat/bran or clay formulations or washed spores resulted in cfu numbers on seeds at levels previously shown to be adequate (>103 cfu per g seed) to control seed-borne diseases in other species. Microscopy of the fluorescent gfp-transformant of IK726 revealed that mycelial growth could be observed particularly on the testa of the seed, including where the radicle emerges during germination.

The effect of the antagonists was investigated in three field experiments.

In the first year, the four antagonists were compared and in the second year different application methods were tested on IK 726 and TRI003. In the third year, treatment with IK726 was combined with tests of two seed lots and two periods of stratification. Also in the second year, a parallel growth chamber trial was made on control of Pythium ultimum var ultimum artificially inoculated soil.

The results from these experiments showed that TRI003 and C. rosea IK726, improved germination in the field (statistically significant) in more than one season. In the first field experiment (2002), where seed coating was just before sowing, TRI003 was the best treatment at the first assessment resulting in 23% germination compared to 19% in the untreated control. In 2003 treatments, coating before stratification resulted in 100% improved germination i.e. from 10% in control to 23% and 22% for TRI003 and IK 726, respectively at the first assessment. At the second assessment, IK726 coated with clay before stratification resulted in 42% healthy plants compared to 27% in the control. In the 3rd year of the experiments, only a vital seed lot that had been sub-optimally stratified was statistically improved with respect to germination percentage by IK726 treatment before stratification, resulting in a germination of 26% healthy plants compared to 19% in control. The weather varied greatly from year to year, with the spring of 2004 being particularly cold.

The growth chamber experiment with artificially inoculated Pythium ultimum confirmed that clay formulated TRI003 and IK726 improved healthy plant stand when seeds were treated pre-stratification.

It can be concluded that

the biocontrol agents TRI003 (Trichoderma harzianum) and Clonostachys rosea IK726 can improve germination in Abies nordmanniana seed beds.
C. rosea IK726 can control seed-borne pathogens as well as soil-borne Pythium ultimum and TRI003 has an effect against this soil-borne pathogen
In the field the highest number of germinated plants was registered when the time of germination was short, due to either use of high-vigour seeds or optimal field conditions. However, antagonist treatment resulted in improved germination of vigorous seeds under suboptimal conditions e.g high infection potential, sub-optimal length of stratification or adverse field conditions.

Overall, we conclude that the practical use of biological control in forest nurseries is pre-mature but the perspectives for further development of biocontrol integrated in a seed technology including stratification are good.

This report also covers work done on seed density and soil texture. Different levels of seed sown per m² and application of sand in the field before sowing were investigated but no beneficial effects could be demonstrated from these measures.