A Practical and Low Cost Microbiotest to Assess the Phytotoxic Potential of Growing Media and Soil

For routine toxicity testing of composts and growing media, two different types of assays are usually applied. One approach is the use of growing trials in pots with various mixtures of the material. In extract-based tests, seeds are germinated in petri dishes and exposed to an exudate from the material to be tested. In this study, one of these methods, the Phytotoxkit microbiotest, was compared to the standard phytotoxicity test with Lactuca sativa as used by the RHP foundation. The Phytotoxkit test was performed in transparent test containers which allow for direct observation and length measurements of the seedlings by means of image analysis. Two dicotyles (Lepidium sativum and Sinapis alba) and one monocotyle (Sorghum saccharatum) were used. When measuring a presumably phytotoxic bark, in comparison with a reference bark, the Phytotoxkit showed a seedling length inhibition of 60–80% for the dicotyles and less than 20% for the monocotyle after three days. The RHP growing trial showed 30% weight inhibition of the lettuce after two weeks of plant growth when comparing the same barks in mixtures. This preliminary comparison showed that the Phytotoxkit microbiotest is a quick and practical bioassay with a high resolution which has a potential to become an international standard. INTRODUCTION Testing growing media and soil improvers on the presence of harmful components is important for quality control and risk reduction in the process of producing potting soils and container media. In liability cases, prior phytotoxicity testing of all the components used in the production of container media is often regarded as a prerequisite to prove careful production. Phytotoxicity tests may involve growing trials, derived from methods used for potting soil and peat (Verhagen, 2000; Morel and Guillemain, 2004). In such tests, a material is mixed in various dilutions with e.g., peat and used in a randomized design container culture trial. Generally two or three plant species from different families are tested, because plants differ widely in their response to different toxins (Wang, 1991). These growing trials are regarded as reliable, but have some disadvantages. They are relatively time consuming, because several weeks to months may be involved. Also, the structure of the growing medium used may influence the plant’s response, and poor structure may be mistaken for toxicity. The mixing of the material with substances as peat results in dilution of the toxin in the root zone. Furthermore, the experiments may start with a plantlet grown in another medium than the mixture tested, which creates chance on interaction. The comparison of results is sometimes difficult, because light levels, temperature and humidity may vary from test to test. Another approach to test for phytotoxicity uses seedling shoot growth in a petri dish as a parameter for toxicity (Fig. 1). These tests are derived from methods for soils, composts and growing media (US EPA, 1996; Baudo et al., 1999; ISTA, 2005). In these tests, some 10 seeds in a few replicates are germinated in petri dishes on wetted filter paper, and exposed to an exudate subtracted from the medium to be tested. After a few days incubation period, the seedlings are stretched out for manual length measurement. Proc. IS on Growing Media Ed.: J.-C. Michel Acta Hort. 779, ISHS 2008 368 These tests are highly sensitive, because of the plants seedling stage. They are generally regarded as reliable and fairly rapid. Disadvantages are the time required for measurements, the water supply and precision. As the seedlings will try to grow vertically in the petri dish, they will become twisted (Fig. 2). The subsequent stretching of the shoot for measurement is time consuming. Furthermore, care must be taken that the water uptake during the germination process is not limited by the maximum amount of water present in the filter paper. The precision of the petri dish assay is interand intralaboratory dependent, as shown by several intercalibration exercises (Beltrami et al., 2002). It is clear that for assessing the phytotoxic potential of growing media and soil, each of the two approaches has specific advantages and weaknesses. Therefore, a new bioassay, the Phytotoxkit (Phytotoxkit, 2004) was tested. This test is performed in flat transparent containers which only allow seedling growth in one direction (Fig. 3). The seeds are on top of a filter paper which is placed on the wetted substrate. The test containers are incubated in vertical position to allow for normal seedling development. After the incubation period, seedling growth can be recorded with a digital camera, which allows for subsequent measurement by image analysis. In order to evaluate the potential of the Phytotoxkit, we compared this bioassay with a standard growing test (Verhagen, 2000). MATERIAL AND METHODS Standard RHP Phytotoxicity Test with Lettuce As a reference 15 L of a mixture was made. The on e component was 75% v/v of a white peat used as standard in the laboratory involved. The other component was 25% v/v of a known non toxic reference bark from the laboratory store. Another mixture of 15 L was made of the standard white peat, 75% v/v, with 25 v% of a presumably phytotoxic bark material, PPBM. The mixtures were prepared under standardised compaction, and mixed with powdered calcium carbonate to reach a pH between 5.0 and 5.5. Fertilizer was added in the form of 0.75 g of PG-mix per litre. Water (EC < 0.1 dS.m) was added to a content of approximately 50% v/v (showing water release on pressing the sample by hand). The mixtures were stored for at least 24 hours at 20°C in the dark, in closed plastic bags. The test was carried out with lettuce (Lactuca sativa). 1. Test Procedure. Per mixture, four replicate sample trays were filled (surface area 0.075 m each). Per tray, 3.0 L of the mixture and 500 ml of water were added. Each sample tray was slightly compressed as prescribed and 3 rows of ten holes, 5 mm depth, were made in the flattened substrate. Each tray was then sowed with 30 lettuce seeds. The trays were stored for 18–24 hours at 12°C. After that period the trays were placed in a plastic tunnel in a greenhouse at 22/20°C (day/night) and RH > 80%. Up to 14 DAS (Days After Sowing) the trays were remoistened every two days. At 7 DAS, germination was scored positive if two lobes were visible; at 14 DAS the above ground fresh weight and the number of plants per tray were measured. Growth depression was reported as ((APWref – APWtest)/APWref) * 100), APW being the average plant weight which is total plant weight divided by number of plants. In formula APWtest = TPWtest / Ntest and APWref = TPWref / Nref, TPW being the total weigh of all plants and N the number of plants weighed. Phytotoxkit Microbiotest The Phytotoxkit tests (MicroBioTests Inc.) were performed according to the Standard Operational Procedure of this assay (Phytotoxkit, 2004) and with the materials included in the commercial Phytotoxkit. The materials to be tested were a presumably phytotoxic bark material and a known non toxic reference bark from the laboratory store. Both materials were used to fill a 1200 ml beaker. Dry weight was established before slow submergence in demineralised water. After 24 hours at 20°C, the material was allowed to drain to equilibrium on a grid and the saturated weight was measured. All