UNIVERSITY OF PANNONIA GEORGIKON FACULTY INSTITUTE FOR PLANT PROTECTION

DOCTORAL SCHOOL OF CROP PRODUCTION AND HORTICULTURAL SCIENCES

HEAD OF SCHOOL

RICHARD GÁBORJÁNYI, professor

INVESTIGATION OF GERMINATION BIOLOGY AND COMPETITIVE ABILITY OF VELVETLEAF ( MEDIC.)

THESIS OF DOCTORAL (PHD) DISSERTATION

WRITTEN BY VIKTOR NAGY

LEADING PROFESSOR ERZSÉBET NÁDASY Associate Professor

KESZTHELY 2012 PRELIMINARIES AND AIMS OF THE RESEARCH WORK

In the last 50 years some weed species, for example velvetleaf (Abutilon theophrasti Medicus) has reached a very high, 10-200-fold cover growth compared to the data of the first national weed survey in our country. Their accumulation is due to climatic causes, taking into account other circumstances as well. Their reproductive strategy, their biology also help them to settle and continuously cause damage on a large part of our fields. According to the data of the fourth national weed survey, velvetleaf was the 27th most important weed on the weed list, but it reached the 15th place by 2007-2008 when the fifth survey was done. The protection against it is almost exclusively based on chemical plant protection at the moment, however since there is an increasing demand for more environmentally friendly plant protection, biological methods can come into view. For that reason, the aim of the present thesis was to study velvetleaf (Abutilon theophrasti Medic.) which has become important in recent years, its biology and the possibility of biological control against it. The content and the topics of my thesis are the following: - Examination of the effect of sowing depth and fertilizers on germination and growth of velvetleaf. - Examination of the allelopathic effect of velvetleaf in laboratory and greenhouse bioassays. - Study of the phytochemical character of velvetleaf, determination of the possible allelochemicals. - Study of the competitive ability of velvetleaf in open-field, small-parcel, additive experiments. - Survey of the pests of velvetleaf.

MATERIALS AND METHODS

Germination biological examinations

We set a pot-experiment afield at the Plant Protection Institute of Georgikon Faculty, Pannon University in 2009 and 2010 to study the effect of sowing depth on the germination and growth of velvetleaf. We performed the experiments in 50-litre pots, we repeated the examinations 4 times, each time we sowed 50 seeds. We collected the seeds at a corn culture in Keszthely, in October, 2008 and after purification we stored them at -15 oC, in a deep freezer in the weed seed collection of Plant Protection Institute until they were used. We put Ramman’s brown forest soil from Keszthely in the pots. First we laid soil in the pots and we placed the seeds uniformly on the leveled ground, then we covered them with a desired height of soil layer. We examined the effects of ten different sowing depths (1, 2, 3, 4, 5, 6, 7, 8, 10 and 15 centimetres) on the germination percentage of velvetleaf and the different growth parameters. We performed assessments in three different sampling times (5, 7 and 9 weeks after sowing). In the course of the occasions of samplings we examined 5 plants each time from the first 5 sowing depths. Plant entities needed to perform the examinations did not come up from deeper than that. During evaluation we considered germination percentage and sowing percentage identical. We measured the shoot length, the weight of fresh and dry shoots and roots, the leaf area and the number of leaves.

We tested the independent and combined effects of three macro nutrition (nitrogen, phosphorus and potassium) fertilizers on the germination and growth of velvetleaf in a greenhouse pot-experiment. We performed our experiments in 1-litre pots, we repeated the tests four times, we sowed 25 seeds each time. We set the experiments on 5th May both years. We put 314 cubic centimetres (approximately 1 kg) air-dried soil and we measured the appropriate dose for field from the three applied fertilizers of different agents that contented macro nutritients. We used Ramann’s brown forest soil for the experiment. The fertilizers and their dose that we used were the following: Linzer NAC [2,325g (200 kg/ha N)], Szuperfoszfát [1,53g (100 kg/ha

P2O5)] és Patentkáli [1,05g (100 kg/ha K2O)]. We desiccated the fertilizers with an electric grinder before use and we mixed them in the soil of the pots. The applied treatments were the following: control, N, NP, NPK, P, K, PK, NK. After sowing we noted the time required for the plants to emerge, we determined germination percentage on the second week after sowing. Four and six weeks after we had set the experiment, we removed 5 plants from the pots. We measured the height and the leaf area of the plants and the weight of the fresh and the dry plant. During the second evaluation we measured the weight of fresh and dry shoots and roots. We determined the nitrogen content of the shoots with Kjeldahl-method. We applied spectrophotometric procedure to define phosphorus content. We specified potassium and calcium content with flame photometer. We deduced the effect on germination and growth of the given, independently or combinedly applied fertilizers from the measured parameters.

Examination on allelopathy

We examined the allelopathic effect of velvetleaf plants on the germination and growth of cereal crops in a laboratory bioassay in the year of 2010. We set the experiments in the laboratory of the Plant Protection Institute of Georgikon Faculty, at Pannon University in September and October, 2010. We tested the allelopathic effect of aqueous, acetone and ethanol extracts made of velvetleaf. We used the air-dried above-ground parts (air-dried leafy stems) and the air-dried roots of velvetleaf in a concentration of 2,5 and 5g/100 ml solvent to prepare the extracts. We chopped the parts of the plants into fine granule size, then after adding the needed extractant and 24 hours of extraction, soaking we filtered the extracts through filter paper and used them immediately. We used cereal crops test plants for our experiments. We studied the effects of the extracts on the germination and growth reactions of five cereal crops [winter wheat (Mv- Magdaléna), winter barley (Cartel), spring barley (KH-Lilla), spring oats (Mv-Pehely), corn (PR35F38)]. When we were choosing the test species, we took cereal crop and corn rotation into consideration that is very common in our country. Because of the rotation, test plants can meet the aqueous extract of velvetleaf plant remains during germination and the phase of early development in field conditions. Only those species were involved in the evaluation that had a 50% germination percentage in case of every treatment. That way, we assessed the growth reactions of winter wheat, corn and spring oats. We performed our experiments in 15 cm diameter Petri dishes, we repeated the tests 4 times, every time we examined 25 seeds on wrinkled filter paper, we dosed 15 ml extract, in case of the control 15 ml tap water per Petri dish. We evaporated the organic solvent extracts after putting them on filter paper, then we attenuated them with 15 ml tap water. We performed germination in a WTB-Binder KB 115 cooling incubator, at 20oC, in dark. We evaluated the experiments ten days after the setting. We examined the germination percentage and measured the length of primary roots and shoots. We were looking for relations between the germination percentage and the lenght of shoots and primary roots.

We set a greenhouse pot experiment at the Plant Protection Institute of Georgikon Faculty, Pannon University in 2010 to study the allelopathic effect of plant remains of velvetleaf mixed in the soil. We performed our examinations between 23rd August and 18th October, 2010. We measured 1 kg air-dried Ramann’s brown forest soil in pots of 2-litres capacity. We mixed dried, leafy parts of stems and dried roots to the soil. We used 50g (5%) or 100g (10%) leafy parts of stems and 25g (2,5%) or 50g (5%) root remains. After we prepared the mixture of soil and plant remains, we immediately sowed the seeds of the test plants. We repeated the examinations 4 times, each time we sowed 30 seeds, then we thinned them to have the same number of stems (15 plants/pot). We used cereal crops test plants for our experiments. We examined the germination and growth reactions of five cereal crops [winter wheat (Mv-Magdaléna), winter barley (Cartel), spring barley (KH-Lilla), spring oats (Mv-Pehely), corn (PR35F38)]. Again, only those species were involved in the evaluation that reached a 50% germination percentage in every pot. That way, similarly to the previous experiment, winter wheat, corn and spring oats were evaluated. In the course of the evaluation, we removed 5 test plants from each pot four and eight weeks after sowing (which took place on 23rd August). We measured the lenght of the shoots and roots of the test plants, their fresh and dry weight and leaf area.

We collected Abutilon theophrasti plant samples at a barley-stubble in Vörs in 2009, the samples were of secondary emergence and 6-8 leaved for their phenological development stage. We could subtract volatile components only in a low amount from the brown-coloured samples (it is an appropriate substance for senescent plants) containing leaves and parts of stems with the above mentioned method (Héthelyi et al., 2009). According to our previous experience, we agreed that the most usable method is SPME (Solid-phase Microextraction) for the instrumental analytical demonstration. Accordingly, we applied GC-MS method that is connected to the SPME CombiPAL automatic sample-rationing system. Instead of 50-100 g sample, 0.2-0.5 g dried plant sample was enough, we did not have to extract volatile oil in a traditional way. We incubated the sample on 60ºC for ten minutes in a closed 20ml glass phial in a fully automathic system and we found that aroma molecules, saturated in the air, absorbe on the Fiber (SPME strand). Next the automathic system relocates the strand into the injector of the gas chromatograph set. In the injector, the sample separates from the strand in one minute with thermal desorption, the injection port evaporates it on 250ºC. From this point the GC/MS measuring continues as usual until the strand can be cleaned in a special, heated cleaning chamber that is streamed by inert gas. The instrument is an AGILENT GC/MS mass spectrometer combined with CombiPAL automatic preparation system (Semmelweis University, Department of Pharmacognosy).

At first, we analyzed the phenoloids and phenol-acid components of seeds of velvetleaf with Thin layer chromatography (TLC). We chopped rough the seeds collected in Keszthely in 2009, then after extracting methanol we poured 10 µl amount on Merck silica gel applied on aluminium foil. The tests: rutoside (=rutin), hiperoside (both of them are yellow- coloured) and chlorogenic acid (blue-coloured). We defined the total amount of tannin in the seeds according to Ph.Hg. VIII. (The eighth edition of the Hungarian Medicament Book published by the National Institute of Pharmacy). We collected samples of velvetleaf from five different phenological development stages (3-5 leaved, 6-8 leaved, 10-12 leaved plants before flowering, during flowering and during ripening) in Keszthely in 2010. We isolated the root and the parts of stem, then we dried the leafy shoots and the root samples on room temperature after chopping them rough. In the case of these samples we also examined them with TLC method, we poured 5 µl amount on Merck silica gel applied on aluminium foil. We assessed TLC results according to so-called Retention value. We measured developed patch distance and distance travelled by the solvent (solvent front) from the place of application or starting point. The quotient of these two items is Retention value or Rf, with the help of which we worked out the relative (%) amount of the components detected in the plant. Investigation on competitive ability of velvetleaf

The aim of our experiment was to measure the competition ability of velvetleaf (Abutilon theophrasti) in parsley (Petroselinum crispum Mill.) culture. The open-field, small parcel additive experiment (cca. 200 parsley plant parcel-1) was set up in Hungary, Békés county, in the field of Antal és Társa Ltd., Apátfalva, in 2010. The study field had a slightly alkalide chernozem soil, with good humus supply. The experiment was carried out in cultivar ’Makói hosszú’, sown in rectangular bed, after autumn wheat forecrop in 3m2 sized parcels in 4 replications. Three different velvetleaf densities were adjusted (2, 5 and 10 velvetleaf plant m-2) besides the weedless control. During the period of the experiment handmade hoeing was managed to exclude other weed plants. The fresh stem and root weight of the crop was measured in each parcel. From each parcel weight and length of 10 randomly choosed root was also measured. The root yield were classified, where two classes were made: first class and processing root (unclassified) and the rate of classes were determined per parcel.

Study of the pest community of velvetleaf

The velvetleaf pests survey was conducted in 2008 and 2009. We were looking for velvetleaf plants for seed collecting in the corn stocks in the fields in Keszthely in September and the first decade of October, 2008. Our secondary aspect was to assess and define any pests that may occur on the plant. During and after seed collection we evaluated visually the species present and their rate of occurance and collected entities of different species by thinning them out for further identification. We could not net the plants because they created a common stock with the corn and it was not possible to use the method because of the size of the plants. In 2009, we investigated velvetleaf damaging agents in the fields and in open-air experiments in culture vessels, too. We set an open-air experiment in 50-litre culture vessels on 29 April, 2009, our main experimental goal was to examine the impact of seed depth on germination and growth. In addition, it was a great opportunity for the continuous monitoring of pest visitors of the plants. The observations were carried out from May to the end of September, we collected the species actually occuring on the plant, we identified them on the basis of the visual damage they made, later we observed and defined them by their species. We assessed the pests in the experiments in culture vessels to refine and expand the results of the observations carried out in the previous year. In Vörs, in September, 2009 young, non-flowering plants were collected from secondary emergence on a barley stubble. We used nets to collect the weed associations that are composing a thick stock (95% of which was velvetleaf being the main stock component), we determined the collected items according to their species and evaluated the possibility to use them for potential biological control according to their entity number and the degree of the damage they made.

NEW SCIENTIFIC RESULTS

1. If we increase sowing depth, it will decrease the germination, the shoot length, the weight of fresh and dry shoots, the leaf area and the number of leaves of velvetleaf, but it will not influence the weight of fresh and dry roots. We determined that 1 centimetre increase in the sowing depth decreases the germination of velvetleaf by 4,5%. It can spring even from 15 centimetres depth on Ramann’s brown forest soil.

2. Nitrogen with a dose of 200kg N/ha reduced the germination, the shoot length, the weight of fresh and dry shoots, the leaf area of velvetleaf. Phosporus and potassium with a dose of 100 kg agent/ha increase germination and growth. The treatments did not influence the nitrogen, phosporus, potassium and calcium content of shoots.

3. The tap-watered, 5% extract made of the dry shoots of velvetleaf decreased the germination and the growth of the shoots and roots of winter wheat (Mv Magdaléna), corn (PR35F38) and spring oats (Mv Pehely). Tap-watered extracts proved to be more effective than acetone and ethanol extracts. The alteration of germination percentage did not influence the length of shoots and roots.

4. Leafy stem pickings of velvetleaf that were mixed in the soil with a dose of 100g/kg of soil hindered the shoot and root progression of the test plants. The dry stem pickings with a dose of 50g/kg had an impeding effect at first, but later they had a stimulating effect. Velvetleaf root mixed in the soil has a stimulating effect on the progression of test plants.

5. During SPME-GC/MS analysis, we identified 23 volatile components from the shoots of velvetleaf. Hexanal, benzaldehyde, benzil-acetaldehyde, limonene, trimethyl- cyslopentanone, ß-cubebene and safranal occured. In thin-layer chromatography tests, we could not identify flavonoid from the seeds, the tannin content of the seed was also low (0,14%). Chlorogenic acid or any other fenol carbon acid were not specific for leafy shoots. We identified rutoside in the shoots, the most of it could be found in 6-8 leaved stems (0,142%).

6. If we increase velvetleaf plant number, the quantity of root crops and the average lenght and weight of roots of parsley will decrease. If we increase velvetleaf plant number by 1 piece/square m, the yield of the parcels decreases by 0,22 kg. The increase in the plant number decreases the quantity of first class roots and increases the quantity of branching, industrial quality roots. The increase in the entity numbers of velvetleaf did not influence the shoot weight and the nitrogen, phosporus, potassium and calcium content of shoots and roots of parsley.

7. We identified 7 and 1 mollusc pest species on velvetleaf. We broadened the literature data with four new pests: (Helix pomatia) edible snail, (Oxycarenus lavaterae) mallow seed bug, parumpunctatus and (Mamestra brassicae) cabbage moth. hyalinus can play an important role in biological protection.