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Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz Journal of Plant Diseases and Protection 112 (4), 343–350, 2005, ISSN 0340-8159 © Eugen Ulmer KG, Stuttgart

Effects of Trichoderma spp. and Gliocladium roseum culture filtrates on seed germination of vegetables and maize Wirkung von Kulturfiltraten von Trichoderma spp. und Gliocladium roseum auf die Keimung der Samen von Gemüsepflanzen und Mais F. Celar*, N. Valic University of Ljubljana, Biotechnical Faculty, Agronomy Department, Institute of Phytomedicine, Jamnikarjeva 101, 1000 Ljubljana, Slovenia * Corresponding author, e-mail [email protected] Received 31 January 2005; accepted 1 April 2005

Summary The subject of this study was to establish whether antagonistic fungi in vitro produce extracellular growth-regulating substances independently of plant presence. The results showed that the only culture filtrate which had no influence on seed germination was that of Gliocladium roseum. The filtrate of Trichoderma koningii had a pronounced negative effect on the first and on the final count of germination of onion, chicory and lettuce seeds. The filtrates of T. longibrachiatum and T. viride had a statistically significant negative effect on the initial germination as well as the final count of germinated onion seeds. The filtrates of T. longibrachiatum, T. harzianum and T. viride had a favourable influence on the first count of germination of spinach seeds. The filtrates of T. longibrachiatum and T. harzianum stimulated the germination of red beet and chicory or tomato and chicory seeds, respectively. In most cases the filtrates only influenced the speed of germination but had no effect on the final number of germinated seeds. The results indicate that the isolates of the antagonistic fungi studied in the experiments produced in vitro growth-regulating substances independently of the plant presence. To determine the nature of these substances further studies should be carried out. Key words:

cabbage; carrot; chicory; Gliocladium roseum; lettuce; maize; onion; pea; radish; red beet; seed germination; spinach; tomato Trichoderma harzianum; Trichoderma koningii; Trichoderma longibrachiatum; Trichoderma viride

Zusammenfassung Es wurde untersucht, ob antagonistische Pilze in vitro extrazelluläre Substanzen zur Wachstumsregulierung in Abwesenheit von Pflanzen bilden können. Die Resultate zeigen, dass nur das Kulturfiltrat von Gliocladium roseum keine Wirkung auf die Keimung der Samen der untersuchten Pflanzen hatte. Das Kulturfiltrat von Trichoderma koningii wirkte ausgesprochen negativ auf die Keimgeschwindigkeit und die Anzahl gekeimter Samen von Zwiebeln, Radicchio und Salat. Die Kulturfiltrate von T. longibrachiatum und T. viride hatten einen statistisch signifikanten, negativen Einfluss auf die Keimung von Zwiebelsamen. Die Kulturfiltrate von T. longibrachiatum, T. harzianum und T. viride stimulierten die Keimung von Spinatsamen. Die Kulturfiltrate von T. longibrachiatum und T. harzianum erhöhten die Keimrate von Roter Bete und Radicchio bzw. Tomate und Radicchio. In den meisten Versuchen wirkten die Kulturfiltrate nur auf die Keimgeschwindigkeit, hatten aber keinen Einfluss auf die Keimfähigkeit bzw. Anzahl gekeimter Samen. Die Ergebnisse deuten darauf hin, dass die hier untersuchten Isolate der antagonistischen Pilze in vitro Substanzen zur Wachstumsregulierung unab-

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hängig von der Präsenz der Pflanzen bilden. Zur Ermittlung der Natur dieser Substanzen sind weitere Untersuchungen notwendig. Stichwörter: Erbse; Gliocladium roseum; Keimfähigkeit der Samen; Kohl; Mais; Möhre; Radicchio;

Rettich; Rote Bete; Salat; Spinat; Tomate; Trichoderma harzianum; Trichoderma koningii; Trichoderma longibrachiatum; Trichoderma viride; Zwiebel

1

Introduction

Brown (1974) and Suslow (1982) studied potential mechanisms developed to explain the effects of growth-promoting microorganisms on higher plants. Numerous speculative explanations of mechanisms involved in increased growth responses have been developed. Growth responses induced by microorganisms could result from inhibition and alteration of the normal root microflora, especially ‘minor pathogens’. Beside direct effects of growth-stimulating substances (hormones and other growth factors), stimulation of nutrient uptake and nutrient availability could also be involved. It is also possible that low levels of substances in soil inhibitory to plant growth could be decreased in concentration by growth-stimulating microorganisms. Strains of fungi belonging to the genus Trichoderma have been used in various biocontrol experiments and reported to be capable of increasing plant growth, even in the absence of pathogens. Among the responses to application of Trichoderma spp. are a faster germination and increases in percentage of emergency, plant height, leaf area and dry weight (Lindsey and Baker 1967, Baker et al. 1984, Windham et al. 1986, Chang et al. 1986, Paulitz et al. 1986, Kleifeld and Chet 1992, Inbar et al. 1996). In soil inoculated with Trichoderma harzianum earlier flowering of periwinkle and increases in the number of blooms per plant on chrysanthemums were reported (Chang et al. 1986). The aim of this research was to determine whether antagonistic strains of Trichoderma spp. and Gliocladium roseum secrete in vitro growth-regulating substances independently of the plant presence. This was approached by studying the effect of the culture filtrates of these fungi on seed germination of different cultivated plants.

2

Materials and methods

2.1

Fungal isolates

The antagonistic fungi used in this study (Table 1) originated from the Mycological Collection of the National Institute of Chemistry, Slovenia (these are marked with B-), or from the Mycological Collection of the Institute of Phytomedicine. All antagonistic fungi in question had previously shown in vitro and in greenhouse experiments antagonistic activities against different phytopathogenic fungi, e. g. Rhizoctonia solani, Sclerotinia sclerotiorum and Fusarim spp. (Ma·ek and Celar 1990, Celar 1992). 2.2

Nutrient media

Two media were used. The first was liquid synthetic medium (LSM) with concentration in g/l: MgSO4 · 7H20, 0.2; NH4NO3, 1.0; K2HPO4, 0.9; KCl, 0.15; FeCl2, 0.002; ZnSO4, 0.002; glucose, 5.0; and distilled water ad 1000 ml. The second, solid synthetic medium (SM) was the same as LSM with the addition of 20 g agar (Ordentlich et al. 1991). 2.3

Disinfection of seeds

The seeds used (Table 2) were disinfected using a 5 % solution of sodium hypochlorite (0.65 % of active chlo-

Table 1.

Isolates of antagonistic fungi used to test the effects of culture filtrates on seed germination of vegetables and maize

Species

Isolate

Trichoderma longibrachiatum Rifai Trichoderma harzianum Rifai Trichoderma viride Pers.: Fr. Trichoderma koningii Oudem. Gliocladium roseum Banier

TL-9A TH-39 B-117 B-123 B-111

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Plants used to test the effects of culture filtrates on seed germination of vegetables and maize and dates at which germination rates were determined

Common name

Latin name

Family

Cultivar

Date*

Onion

Allium cepa L.

Liliaceae

‘Rossa di Lucca’

6/12

Radish

Raphanus sativus L. var. sativus

Brassicaceae

‘Non Plus Ultra’

4/14

Spinach

Spinacia oleracea L.

Chenopodiaceae

‘Meraviglia mercato’

7/21

Carrot

Daucus carota L.

Apiaceae

‘Berlicum’

7/14

Tomato

Lycopersicon esculentum Mill.

Solanaceae

‘St. Pierre’

5/14

Cabbage

Brassica oleracea L. convar. capitata (L.) Alef. var. capitata L.

Brassicaceae

‘Kranjsko okroglo’

5/10

Pea

Pisum sativum L. ssp. sativum

Fabaceae

‘Petit Provencal’

5/8

Red beet

Beta vulgaris L. ssp. vulgaris var. vulgaris

Chenopodiaceae

‘Egiptian Turnip ‘Rooted’

4/14

Chicory

Cichorium intybus L. var. foliosum Hegi

Asteraceae

‘Pan di zuccero’

5/14

Lettuce

Lactuca sativa L.

Asteraceae

‘May Queen’

4/7

Maize

Zea mays L.

Poaceae

‘Lj – 180’

4/7

* Date of first and final count (days after placement of seeds on blotters).

rine), to which 0.1 % of Tween was added (to reduce the surface tension). The seeds were immersed into the solution for five minutes and then thoroughly rinsed with sterile distilled water and dried. The effect of the disinfection was checked by transferring the samples of the treated seeds to Petri dishes with sterile solid growing medium (1.5 % water agar) and incubated at 25 °C. The Petri dishes were checked after five days for possible infection. In the case of infection, the time of disinfection with sodium hypochlorite was prolonged or the seeds were immersed into 95 % ethanol prior to treatment (this was necessary especially for those types of seeds with rough surface, namely carrot, tomato and red beet). 2.4

Preparation of culture filtrates and performance of germination assays

LSM (100 ml) was sterilized in 300 ml Erlenmeyer flasks, cooled to room temperature and inoculated with three pieces (5 mm in diameter) of seven to ten days old mycelium of each fungus previously grown on SM at 25 °C. After inoculation, the Erlenmeyer flasks were incubated in a shaking water bath (25 °C and 120 rpm) for 10 days. After this period the cultures were filtered by suction through three layers of filter paper so that the mycelium was removed. The filtrates were freeze-dried and stored in a freezer until use. Before use, 20 ml of distilled water were added to the lyophilisates and the resulting solution was passed through a membrane filter (Sartorius, 0.2 µm). A sterile blotting paper in a Petri dish was then appropriately moistened with the lyophilisate. In the control treatment sterile distilled water was used. Seeds of the examined plants, previously disinfected as described above, were placed on the blotting paper at 20–100 (depending on the species) per plate. The experiments were carried out in four replications with 100 seeds each per species. The Petri dishes were then sealed with parafilm and incubated in a growth chamber at 20 °C and 80 % relative humidity. After standardised incubation periods (depending on plant species; compare Table 2) a first and a second (final) count of the number of germinated seeds was made (International Rules for Seed Testing, 2004). Due to a high number of seeds per replication, high homogeneity of the material (seeds) used in the experiment and especially the fact that the data was not normally distributed (prerequisite for ANOVA), Z test (α = 0,01) was used for statistical evaluation (Zar 1999). The experiment was repeated twice. The data presented in the tables are the means of two experiments with four replications à 100 seeds.

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Results

Compared to the control treatment, culture filtrate of T. longibrachiatum significantly reduced the germination rate of onion seeds at both counting dates. On the other hand, the same culture filtrate significantly stimulated the initial germination of spinach, red beet and chicory. The effect was, however, transient and not visible anymore at the second counting date (Table 3). Similarly, the culture filtrate of T. harzianum stimulated the initial germination of seeds of spinach, tomato and chicory, but had no influence on the final number of germinated seeds (Table 4). Culture filtrate of T. viride significantly inhibited the germination of onion seeds (first and final count) but significantly stimulated the germination of spinach seeds (first count only) (Table 5). Culture filtrate of T. koningii significantly inhibited the germination of onion, chicory and lettuce, which was obvious at the first and the final count. It also tended to inhibit the germination of most other plant species, but, compared to the control treatment, the differences were not statistically significant (Table 6). The filtrate of G. roseum neither stimulated nor inhibited the germination of the seeds of the plants included in the study (Table 7).

4

Discussion

Antagonistic fungi may directly or indirectly influence growth and development of higher plants. In several studies it was established that antagonistic fungi can have stimulating effects on higher plants (Windham et al. 1986, Paulitz et al. 1986, Kleifeld and Chet 1992, Inbar et al. 1996, Chang et al. 1986), although the influence may in some cases be negative as well. However, only limited information is available on negative effects of antagonistic fungi on higher plants. Negative effects were mostly recorded when a large amount of inoculum of an antagonistic fungus was used. For example, inoculation with large amounts of inoculum of Trichoderma spp. partially reduced the germination of sugar beet seeds and inhibited root growth as well. No negative influence was established in cabbage, lettuce and garden cress. This was related to alkyl-pyrones formation, volatile metabolites which can act anti-fungal or can inhibit development of some plant species and have phytotoxic effects (Kohl and Schlösser 1989). Pathogenic isolates of Trichoderma spp. on maize were found by McFadden and Sutton (1975). In tests

Table 3.

Effect of Trichoderma longibrachiatum culture filtrate on germination of different cultivated plants. Values for “control” and “filtrate” of a given cultivar/counting date marked with asterisks are significantly different (Z-test; α = 0,01)

Plant species

Germination (%) First count

Onion Radish Spinach Carrot Tomato Cabbage Pea Red beet Chicory Lettuce Maize

Table 4.

Effect of Trichoderma harzianum culture filtrate on germination of different cultivated plants. Values for “control” and “filtrate” of a given cultivar/counting date marked with asterisks are significantly different (Z-test; α = 0,01)

Plant species

Final count

Control

Filtrate

Control

Filtrate

90* 91 73* 76 78 89 89 91* 77* 76 90

14* 94 81* 80 83 89 89 95* 86* 80 91

93* 95 79 86 90 96 90 93 86 80 90

15* 97 81 85 90 95 89 95 87 81 91

Germination (%) First count

Onion Radish Spinach Carrot Tomato Cabbage Pea Red beet Chicory Lettuce Maize

Final count

Control

Filtrate

Control

Filtrate

90 91 73* 76 78* 89 89 91 77* 76 90

86 94 82* 81 84* 90 90 93 84* 80 89

93 95 79 86 90 96 90 93 86 80 90

93 97 83 84 91 96 90 94 85 80 91

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Effect of Trichoderma viride culture filtrate on germination of different cultivated plants. Values for “control” and “filtrate” of a given cultivar/counting date marked with asterisks are significantly different (Z-test; α = 0,01)

Plant species

Germination (%) First count

Onion Radish Spinach Carrot Tomato Cabbage Pea Red beet Chicory Lettuce Maize

Table 6.

Effect of Trichoderma koningii culture filtrate on germination of different cultivated plants. Values for “control” and “filtrate” of a given cultivar/counting date marked with asterisks are significantly different (Ztest; α = 0.01)

Plant species

Final count

Control

Filtrate

Control

Filtrate

90* 91 73* 76 78 89 89 91 77 76 90

76* 91 81* 79 79 89 90 91 83 80 89

93** 95 79 86 90 96 90 93 86 80 90

79** 96 81 84 88 96 91 91 85 80 90

Germination (%) First count

Onion Radish Spinach Carrot Tomato Cabbage Pea Red beet Chicory Lettuce Maize

347

Final count

Control

Filtrate

Control

Filtrate

90* 91 73 76 78 89 89 91 77* 76* 90

24* 92 69 74 77 84 86 89 38* 48* 87

93** 95 79 86 90 96 90 93 86** 80** 90

28** 96 79 83 89 93 86 91 48** 53** 88

*, ** Statistically significant difference between treatments (control treatment: filtrate).

carried out by Koch (2001) growth promotion Table 7. Effect of Gliocladium roseum culture filtrate and growth inhibition induced by antagonistic fungi on germination of different cultivated plants. were observed. Commercial mycofungicide Soilgard Values for “control” and “filtrate” of a given (Gliocladium virens) reduced germination and seedcultivar/counting date marked with asterling development of lettuce. isks are significantly different (Z-test; α = 0.01) The aim of the study was to determine whether selected isolates of antagonistic fungi in vitro proGermination (%) duce growth-regulating substances, which influ- Plant ence seed germination of different cultivated plants. species First count Final count Tests were performed in Petri dishes on a blotting paper. Plants, as a possible factor influencing proControl Filtrate Control Filtrate duction of stimulatory or inhibitory substances, were excluded. In a majority of previous studies the Onion 90 88 93 90 influence of antagonistic fungi on seed germina- Radish 91 89 95 94 73 75 79 79 tion was established in soil or other substrates, Spinach 76 77 86 85 while in this study only nutrient media were used. Carrot 78 78 90 89 So it is not granted that positive or negative effects Tomato 89 88 96 94 as observed in the experiment on blotting paper Cabbage 89 87 90 88 will also appear on potted plants or in the field. Pea Red beet 91 89 93 90 Substances excreted in tiny amounts may easily be Chicory 77 77 86 86 absorbed to organic or anorganic soil particles, so Lettuce 76 76 80 79 that neither inhibition nor stimulation of growth Maize 90 89 90 89 may result. This question has been at least partially denied by the results of two previous studies. Some of the isolates of antagonistic fungi used in the study, have indicated antagonistic activity against soil phytopathogenic fungi and stimulatory effect on plant growth at the same time, when used in the greenhouse experiments (Ma·ek and Celar 1990, Celar 1992). In the control treatment sterile distilled water was used. However, the real control would be the medium. It is clear that this would be difficult to perform, because it is not known to what extent the

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Percentage of ammonium and nitrate nitrogen form remaining in cultures of antagonistic fungi (after Celar 2003)

Antagonistic fungal species

Trichoderma longibrachiatum T. harzianum T. viride T. koningii Gliocladium roseum

Percentage of nitrogen remaining in the form of NH4+

Percentage of nitrogen remaining in the form of NO3–

Three days

Six days

Three days

Six days

26 55 27 34 44

09 23 01 28 35

100 100 100 100 089

100 100 100 090 069

nutrients are depleted due to growth of the fungus. But on the other hand it is obvious, that the medium is quite different from distilled water. The LSM medium was not appropriate for control due to a high possibility of contamination with saprophytic organisms. Regarding the glucose and nitrogen content, LSM is a relatively poor medium. Trichoderma and Gliocladium are known as fast-growing fungi, therefore the use of nutrients from the medium is rather high. In the previous study it was determined how fast isolates of the used antagonistic fungi utilize nitrogen (ammonium and nitrate form) from the LSM medium. Results are partially shown in Table 8. The ammonium form has been used quickly and after six days only 1–35 % was left in the medium, while the nitrate form remained almost intact (Celar 2003). As described in the methodics, fungi have been reared in the LSM for 10 days and fungi have probably used the most nutrients from the medium. Though it is not quite clear, if the remains of the nitrate form could have influenced the seed germination. Some researches have shown that nitrogen may negatively influence germination and early development in some plants. These experiments confirm that nitrogen in the ammonium form is more inhibitory than nitrate, but it is concluded that it is the elongation of the seedling radicle that is primarily inhibited by ammonium, rather than the seed germination itself. Tolerance to the ammonium ion depends on the plant species too (Westwood and Foy 1999, Muslemanie 1994, Pieterse 1989). Based on extrapolation of the results of Celar (2000), it could be assumed that germination is inhibited or negatively influenced by filtates of the media in which more unutilized nitrogen (especially ammonium form) is left. The results obtained in the study deny this assumption and inhibited germination is not necessarily caused by a higher content of nitrogen remains in the medium. For example, T. harzianum, T. koningii and G. roseum utilize the nitrogen from the medium at an approximately similar pace. Six days after inoculation 23 to 35 % of ammonium form is left in the medium (Table 8). Filtrate of T. harzianum had a stimulatory influence on germination time in some cases but no effect was determined in others. Filtrate of T. koningii had a distinctivly negative influence on germination time in onion, cichory and lettuce, while the filtrate of G. roseum had no influence at all. In this study only the culture filtrate of G. roseum failed to influence the germination of seeds of the tested plants. The filtrate of T. koningii had a distinctly negative influence on the first count as well as on the final count of the germination of seeds of onion, chicory and lettuce. It seems likely that the fungus secretes some toxic substances which inhibit the germination. It has been established that T. koningii produces the metabolite koninginin-A which inhibits the growth of coleoptiles of wheat (Cutler et al. 1989). Culture filtrates of T. longibrachiatum and T. viride significantly reduced the first count and the final count of germination of onion seeds. The germination of spinach seeds was stimulated by the filtrates of T. longibrachiatum, T. harzianum and T. viride. The filtrates of T. longibrachiatum and T. harzianum stimulated the germination of the seeds of red beet and chicory, and tomato and chicory, respectively. In all the cases where a stimulating effect was observed, the culture filtrates had an effect on the first count but did not influence the final count. Windham et al. (1986) also observed that seeds of maize, tomato and tobacco germinated one to two days earlier in the soil inoculated with Trichoderma spp., compared to the control treatment. Seeds of tobacco in a seed-bed germinated much better if T. harzianum had been added into the soil (Cole and Zvenyika 1988) and

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metabolites of the fungus T. harzianum stimulated the germination of black gram seeds (Phaseolus mungo) (Gupta et al. 1995). From the results obtained in our study it can be concluded that in vitro production of growth-regulating substances by the examined strains of antagonistic fungi does not depend on the presence of the plant. Inhibition or stimulation of germination may be related to the substances which regulate the ratio between gibberellins and abscisic acid. These regulate the activity of hydrolytic enzymes, including amylases (Falkowski et al. 1983; Negi et al. 1983). It is also known that some fungi themselves produce gibberellins and other growth-stimulating substances which can affect germination (Kuhad et al. 2004, Windham et al. 1986, Baker et al. 1984). Besides, substances excreted by fungi may directly influence the hydrolytic enzymes (Abd El-Razik et al. 1975). To determine the nature of these substances further studies need to be conducted. The earlier germination of seeds is reflected in the plant height and weight after a certain time. This is of major importance for pot experiments, which usually last a short period of time (three to four weeks). In case the seed germinates two to three days earlier, this represents approximately 10 % of a growing period, which is not negligible and has to be taken into account in the interpretation of the results.

Acknowledgments This work is a part of the programme Horticulture No. P4-0013-0481, granted by the Slovenian Ministry of Higher Education, Science and Technology.

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