Scholarly article on topic 'Suppression of Growth of Fusarium Verticillioides Niren. Using Strains of Trichoderma Harzianum from Maize (Zea Mays) Plant Parts and Its Rhizosphere'

Suppression of Growth of Fusarium Verticillioides Niren. Using Strains of Trichoderma Harzianum from Maize (Zea Mays) Plant Parts and Its Rhizosphere Academic research paper on "Industrial Biotechnology"

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Academic research paper on topic "Suppression of Growth of Fusarium Verticillioides Niren. Using Strains of Trichoderma Harzianum from Maize (Zea Mays) Plant Parts and Its Rhizosphere"

JOURNAL OF PLANT PROTECTION RESEARCH

Vol. 49, No. 4 (2009)

DOI: 10.2478/v10045-009-0072-7

SUPPRESSION OF GROWTH OF FUSARIUM VERTICILLIOIDES NIREN. USING STRAINS OF TRICHODERMA HARZIANUM FROM MAIZE (ZEA MAYS) PLANT PARTS AND ITS RHIZOSPHERE

Ayodele Adegboyega Sobowale1*, Adegboyega Christopher Odebode2 Kitty Frances Cardwell3, Ranajit Bandyopadhyay4

1 Department of Plant Science and Applied Zoology, Olabisi Onabanjo University, P.M.B. 2002, Ago-Iwoye, Ogun State, Nigeria

2 Department of Botany and Microbiology, University of Ibadan, Ibadan, Oyo-State, Nigeria 3800 9th St SW rm 3436, Washington DC 20024, USA kittycardwell@hotmail.com, kcardwell@csrees.usda.gov

4 Pathology Unit, International Institute of Tropical Agriculture, C/o L.W. Lambourn (UK) Ltd, Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK, R.Bandyopadhyay@cgiar.org

Received: January 23, 2009 Accepted: November 26, 2009

Abstract: Three strains of Trichoderma harzianum (strain 1: IMI 380934; strain 2: IMI 380935; strain 3: IMI 380938) were compared for their ability to suppress radial growth of Fusarium verticillioides in vitro. Each Trichoderma strain was paired with the pathogen F. verti-cillioides on 9 cm Petri plates of acidified potato dextrose agar using three pairing methods. Varying growth suppression of pathogen by Trichoderma strains was rated and ratings were analysed using GLM Procedure of SAS. Growth inhibition of F. verticillioides by each of the T. harzianum strains was significantly different from control irrespective of pairing method (p = 0.01, R2 = 0.96). Higher inhibition of F. verticillioides was obtained by inoculating antagonist before pathogen even at p = 0.01. Mode of suppression includes mycopara-sitism and competition for space and nutrients. Growth inhibition of pathogen differed significantly among (p > 0.0001) and within (p > 0.026) pairing methods. T. harzianum strain 1 had better suppression of pathogen than the other two strains when it was inoculated before the pathogen while T. harzianum strain 3 was better when pathogen and antagonist were inoculated simultaneously (p = 0.05). Different strains of T. harzianum could thus be employed as promising antagonists of F. verticillioides.

Key words: antagonist, Fusarium verticillioides, pathogen, Trichoderma harzianum, strain

INTRODUCTION

Among major cereal crops in the world production, maize (Zea mays) was documented to rank third following closely behind wheat and rice (Bunting et al. 1978). It could easily pass as the world's most widely distributed crop with a relatively lower price compared to other cereals. It could thus be said to enjoy wider range of uses than any other cereals (Bunting et al. 1978). Fusarium verticillioides (formerly F. moniliforme) is one important pathogen known to be a constant companion of maize with the ability to cause disease at all stages of maize plant development (Kedera et al. 1992). Apart from maize, it actually infects a wide range of other crops like sorghum, wheat, barley, etc. worldwide (Visconti and Doko 1994; Soonthornpoct et al. 2000). It is not only the most common pathogen of maize, but also the most common fungus colonizing symptomless maize plants (Munkvold and Desjardins 1997). It has been implicated in the stem rot of maize (Zea mays). It is known to be mycotoxigenic and carcinogenic, posing great threats to human and animal health (Marasas 1988; Julian et al. 1995).

Trichoderma is a unique genus that is made up of fungi most commonly used as biocontrol fungi against many pathogens in vitro and in vivo (Paavanen-Huhtala et al. 2000). Mycoparasitism, competition, and antibiosis, amongst others, are different mechanisms by which members of the genus bring about their biocontrol activity (Campbell 1988; Wells 1988; Sharma and Sankaran 1988). Howell (2003) hinged their successful biocontrol records on their ability to parasitize other fungi. Amongst several other reports, T. viride, isolated from roots of maize plants was reported to suppress radial colony extension of F. verticillioides in vitro (Yates et al. 2000). T. harzianum amongst others are also reported to be effective in controlling pigeon pea wilt of Fusarium oxysporum f. sp. udum (Soma-sekhara et al. 1996). The in vitro experiment was a preliminary study to examine effectiveness of T. harzianum as an antagonist of the maize (Z. mays) stem rot pathogen F. verticillioides as well as the effect of pairing method on its antagonistic potential.

""Corresponding address: delesobowale@yahoo.com

MATERIALS AND METHODS

Isolating and identifying T. harzianum and F. verticillioides

Naturally infected maize (Z. mays) stems, brought into the laboratory from the field were split open longitudinally using sharp sterile knife. Fragments from the rotting parts were prepared and surface sterilized in 1% sodium hypochlorite (for 5 minutes) and later rinsed in five separate beakers with sterile distilled water. Sterile forcep was then used to pick up and place the rotted fragments onto sterile filter papers which were wrapped for 5 minutes. Later the dried fragments were plated in Petri plates with acidified potato dextrose agar (APDA). The plates were incubated at 28-30°C for 10 days. Resulting mixed cultures of fungal colonies were subcultured appropriately to obtain pure cultures. To identify F. verticillioides, which forms long chains of conidia on potassium chloride medium (Nelson et al. 1983), each Fusarium isolate was cultured in Petri plates containing potasium chloride (KCl) medium.

The Trichoderma species were isolated from different parts of maize plant and its rhizosphere using stalk sectioning and soil dilution plate method (Tuite 1969). They were sent to International Mycological Institute (IMI) for identification after pure cultures of each fungus was stored on silica gel using the method of Smith and Onions (1983).

Pairing T. harzianum strains with F. verticillioides

Individual strains of T. harzianum (AG) were tried as potential antagonists of pathogen (P) by pairing both fungi in vitro. The two fungi were inoculated at opposite ends of 9cm Petri plate with APDA using three pairing methods. These were: inoculation of antagonist two days before pathogen, AGb4P; inoculation of pathogen two days before antagonist (Pb4AG); and simultaneous inoculation of pathogen and antagonist (AGP) (Sobowale et al. 2005). Three replicates were done for each pairing method. All plates were incubated for 30 days at 28-30°C.

Collecting data and statistical analysis

The radial growth of both pathogen and antagonist were measured daily until growth stopped. Varying extent of growth inhibition of F. verticillioides by T. harzianum strains in Petri plates was estimated using the rating scales of 0-10 viz., 0: if antagonist grows and covers the whole 9 cm plate; 1: if pathogen covers between 0 to 0.90 cm of plate; 2: if pathogen covers between 0.91 to 1.80 cm of plate; 3: if pathogen covers between 1.81 to 2.70 cm of plate; 4: if pathogen covers between 2.71 to 3.60 cm of plate; 5: if pathogen covers between 3.61 to 4.50 cm of plate; 6: if pathogen covers between 4.51 to 5.40 cm of plate; 7: if pathogen covers between 5.41 to 6.30 cm of plate; 8: if pathogen covers between 6.31 to 7.20 cm of plate; 9: if pathogen covers between 7.21 to 8.10 cm of plate; and 10: if pathogen grows between 8.1 cm and the whole 9 cm diameter plate. Resulting values of ratings were analyzed using the General Linear Model option of SAS. This was done to compare relative antagonistic potential of the T. harzianum strains against F. verticillioides

in vitro and to determine pairing method that best aided effective radial growth inhibition of F. verticillioides.

RESULTS

T. harzianum strain 1 (IMI 380934) paired with F. verti-cillioides

After inoculating T. harzianum strain 1 two days before F. verticillioides, contact was made between them within two days of pairing. By the 4th day of pairing, T. harzianum strain 1 grew fast in all Petri plates leaving very little space for growth of pathogen. The pathogen barely had a chance to grow to an average of 1.5 cm diameter. On the 6th day after inoculation, all Petri plates appeared as pure cultures of antagonist (Fig. 1a). There was no zone of inhibition.

After inoculating F. verticillioides two days before T. harzianum strain 1, in one Petri plate, contact was made between the two fungi within two days of pairing. In the other two Petri plates, contact was established within four days of pairing. In all Petri plates, the pathogen was restricted to an average growth of 2.5 cm diameter, though reactions in the three Petri plates differed. In the first Petri plate, sporulation of antagonist seemed delayed till the 7th day of pairing except for the areas around point of inoculation. However in the other two Petri plates, sporulation begun by the 4th day of pairing. By the 6th day of pairing, antagonist started growing over mycelim of pathogen in all the three Petri plates. By the 9th day, mycelium of pathogen started drying up from point of contact with the antagonist backwards. Agar was not coloured and there was no zone of inhibition (Fig. 1b).

After inoculating both fungi simultaneously, antagonist grew fast, terminating growth of pathogen at an average of 3.2 cm diameter. There was no clear contact between pathogen and antagonist in any of the three plates. Some inconspicuous space (5 mm diameter on average) resembling a clear zone was observed in all Petri plates. By the 6th day, antagonist started sporulating heavily and speedily on mycelium of pathogen forming white and green knots thereupon (Fig. 1c). Agar was not coloured in any of the Petri plates while zone of inhibition was observed in some Petri plates.

T. harzianum strain 2 (IMI 380935) paired with F. verti-cillioides

After inoculating F. verticillioides two days after T. har-zianum strain 2, contact was made between them within two days of pairing. Antagonist grew fast round the plates restricting pathogen to its point of inoculation on one plate and to an average of 1.3 cm diameter on the other two plates. By the 8th day of pairing, most Petri plates appeared as pure cultures of antagonist. There was no zone of inhibition and agar was not coloured (Fig. 2a).

After inoculating F. verticillioides two days before T. har-zianum strain 2 however, contact was made between them within three days of pairing. Antagonist grew fast, sporu-lating on pathogen's mycelium, and later halting its growth at an average of 3.7 cm diameter. By the 9th day of pairing, mycelium of pathogen started drying up right from point of contact with antagonist backwards, this was not observed in

Fig. 1. T. harzianum strain 1 (AG3) inoculated before P (F. verticillioides) (a); P inoculated before AG3 (b); AG3 inoculated simultaneously with P (c). AG3 is seen obliterating mycelia mass of P after 20 days of pairing (a). AG3 gradually distorted the mycelia growth of P from point of contact (b), sporulating with time on the entire mycelia mass of P (b & c).

pure culture of pathogen. Agar was not coloured and there was no zone of inhibition in any of the plates (Fig. 2b).

Simultaneous inoculation of both fungi also showed fast growth of antagonist stopping radial growth of pathogen at an average of 2.7 cm, later sporulating upon its mycelium by the 6th day of pairing. Agar was not coloured in any of Petri plates, but a clear zone of 6.5 cm in diameter on average seemed evident in Petri plates. By the 9th day, antagonist sporulated heavily upon entire mycelium of pathogen, which in turn gradually dried up from point of contact with antagonist backwards (Fig. 2c).

In Petri plates where T. harzianum strains 1 and 2 completely overgrew pathogen's mycelium, mycelium of pathogen seemed to be completely 'fed' upon and the plates appeared as pure cultures of antagonists. This was observed by the 11th day of pairing. In plates where the two Trichoderma strains did not completely overgrow pathogen, its mycelium was so distorted in several spots that agar surface could be seen as spots within mycelium of the pathogen. These were areas where mycelium of pathogen was completely 'fed' upon to agar surface. This was observed for both strains of T. harzianum irrespective of pairing method.

AG4 INOC. Ь4 P

Fig. 2. T. harzianum strain 2 (AG4) inoculated before P (F. verticillioides) (a); P inoculated before AG4 (b); AG4 inoculated simultaneously with P (c). AG4 gradually grew on P until the latter was completely obliterated (a). AG4 gradually sporulated on P from point of contact until it overgrew the entire mycelia of P (b & c) after 20 days of pairing.

T. harzianum strain 3 (IMI 380938) paired with F. verticillioides

T. harzianum strain 3 also grew very fast on mycelium of F. verticillioides when inoculated two days before pathogen, stopping growth of F. verticillioides at an average of 0.97 cm diameter. The pathogen (F. verticillioides) had no chance to grow on any of Petri plates (Fig. 3a). When F. verticillioides was inoculated two days before T. harzianum strain 3, still the latter grew fast, making contact with pathogen within two days of pairing, terminating its radial growth at an average of 4.17 cm diameter (Fig. 3b).

Simultaneous inoculation of both fungi still caused antagonist growing fast, making contact with pathogen within two days of pairing, thereby stopping its further growth at an average of 2.13 cm diameter. By the 7th day of pairing, mycelium of pathogen started drying up, the occurrence that was not observed in pure culture of pathogen. By the 10th day of pairing, antagonist sporulated heavily on mycelium of pathogen in most Petri plates so that the plates almost appeared as pure cultures of antagonist (Fig. 3c).

Fig. 3. T. harzianum strain 3 (AG7) inoculated before P (F. verticillioides) (a); P inoculated before AG7 (b); AG7 inoculated simultaneously with P (c). Mycelia of P is seen in 'a' completely overgrown by AG7 after 20 days of pairing.

Suppression of growth of F. verticillioides by the three strains of T. harzianum in all pairing methods

In the analysis of growth suppression of pathogen by antagonists (Table 1), means for growth suppression of F. verticillioides by the three strains of T. harzianum were significantly different from the control (p = 0.01). However, means for growth suppression of F. verticillioides by the three T. harzianum strains were not significantly different from each other (R2 = 0.96). Results of 'inoculating antagonist before pathogen' was significantly different (p = 0.01) from results obtained using other two pairing methods (Table 2). However at p = 0.05 (Table 2), simultaneous inoculation of pathogen and antagonist was also significantly different from inoculating pathogen before antagonist. F value for the pairing methods was highly significant (p > 0.0001) while that for antagonist was not (p > 0.66). Interaction between antagonist and pairing method (p > 0.026) was also significant (Table 3).

Table 1. Comparison of means for radial growth suppression of

F. verticillioides by T. harzianum strains in three pairing methods after 30 days of pairing

Combination Means for radial growth of pathogen

Control 9.67 a

T. harzianum strain 2 3.22 b

T. harzianum strain 1 3.00 b

T. harzianum strain 3 2.89 b

LSD (0.01) 1.03

R2 0.96

Means marked with different letters are significantly different from each other

Table 2. Comparison of growth suppression of F. verticillioides by T. harzianum strains among the pairing methods

Inoculation method Means for radial growth of pathogen LSD (0.01) = 1.03 Means for radial growth of pathogen LSD (0.05) = 0.76

Pathogen inoculated before antagonist (Pb4AG) 4.44 a 4.44 a

Antagonist and pathogen inoculated simultaneously (AGP) 3.44 a 3.44 b

Antagonist inoculated before pathogen (AGb4P) 1.22 b 1.22 c

Means marked with different letters are significantly different from each other

Table 3. ANOVA table for growth suppression of F. verticillioides by T. harzianum strains in three pairing methods

Source DF MS F-Value P > F

Model 11 32.28 53.01 0.0001**

Antagonists 2 0.26 0.43 0.66

Pairing methods 2 24.48 40.21 0.0001**

Antagonist* pairing method 4 2.04 3.35 0.026*

Replicates 2 0.36 0.59 0.56

Error 24 0.61

Total 35 10.54

where:

Antagonists - T. harzianum strains

Antagonist*pairing method - Interaction between antagonist and pairing method

* significant ** highly significant

Suppression of growth of F. verticillioides by the three strains of T. harzianum in separate pairing method

In 'inoculation of antagonist before pathogen' (AGb 4P), means for growth suppression of pathogen by all the T. harzianum strains (Table 4) differed significantly from control (p = 0.05). However means for growth suppression of F. verticillioides by the T. harzianum strains were not significantly different from each other (R2 = 0.27). In 'inoculation of pathogen before antagonist' (Pb4AG), means for growth suppression of F. verticillioides by the three T. harzianum strains were significantly different from control (p = 0.05, R2 = 0.78). Mean for growth inhibition of pathogen by T. harzianum strain 1 also differed significantly from those of strains 2 and 3 which were not significantly different from each other (Table 4).

In 'simultaneous inoculation of pathogen and antagonist' (AGP), means for growth suppression of F. verticillioides by the three strains of T. harzianum also differed significantly from control (p = 0.05, R2 = 0.80) (Table 4). Mean for growth suppression by T. harzianum strain 1 significantly differed from that of T. harzianum strain 3, but not from strain 2.

Table 4. Comparison of means for radial growth suppression of F. verticillioides by T. harzianum strains in separate pairing method after 30 days of pairing

Combination Means for radial growth of pathogen

Control AGb4P Pb4AG AGP

9.67 a 9.67 a 9.67 a

T. harzianum strain 1 1.67 b 3.33 b 4.00 b

T. harzianum strain 2 1.33 b 5.00 c 3.33 bc

T. harzianum strain 3 0.67 b 5.00 c 3.00 c

LSD (0.05) 3.23 1.51 0.76

R2 0.27 0.78 0.80

Means marked with different letters are significantly different from each other

AGb4P - antagonist inoculated before pathogen Pb4AG - pathogen inoculated before antagonist AGP - antagonist inoculated simultaneously with pathogen

DISCUSSION

The ability of the three strains of T. harzianum to significantly inhibit growth of pathogen (F. verticillioides) when compared to control in the three pairing methods supported the submission of many researchers on Tricho-derma species and their antagonistic potential against several fungi (Ahmed et al. 2000; Howell 2003). A major characteristic of the genus Trichoderma, as reported by Howell (2003) is its ability to parasitize other fungi in the course of their antagonistic exploits. The results specifically supported the submission of Ahmed et al. (2000) on the antagonistic potential of T. harzianum. Significant growth inhibition of F. verticillioides by the three T. harzia-num strains over control show their antagonistic ability in vitro just as it was shown in vivo against other fungi by many researchers (Ahmed et al. 2000; Howell 2003; So-bowale et al. 2007). The fast growth of the T. harzianum strains in all Petri plates, irrespective of pairing method with F. verticillioides is indicative of their high sporulat-ing capacity which enabled them to colonize more space on Petri plates, leaving little or sometimes no space for growth of pathogen. A fast and high rate of sporulation is a major characteristic of a good antagonist (Campbell 1988; Sharma and Sankaran 1988).

Mode of inhibition of the pathogen by any of the three T. harzianum strains might include competition for space and nutrients. This was because of the ability of any of them to grow fast round a Petri plate even in the presence of pathogen, irrespective of pairing method, colonizing more space and utilizing the nutrients thereof. The pathogen, in the presence of antagonists, (particularly T. harzia-num strains 2 and 3) seemed to be depleted of essential nutrients, this being suggested by the drying up of my-celial mass of F. verticillioides from point of contact with the antagonists backwards, a phenomenon which was not observed in a pure culture of pathogen. Deacon and Berry (1992) concluded that competition for nutrients might be the commonest mechanism in biocontrol and that other mechanisms only serve as facilitating mechanisms. More

work however needs to be done to ascertain this as a mode of inhibition of the pathogen by Trichoderma strains.

Mode of growth inhibition of pathogen by Trichoderma strains might also include mycoparasitism. This was suggested by a heavy sporulation of the three Trichoderma strains upon mycelial mass of F. verticillioides in all Petri plates, irrespective of pairing method, where some Petri plates appeared as pure cultures of antagonists. The same was also suggested by a complete distortion and subsequent total disappearance of mycelial mass of F. verticillioides (which had grown initially to some points) where the antagonists overgrew them. At such points, re-isolation of F. verticillioides became impossible. Although this is not enough to ascertain mycoparasitism as a mode of antagonism, yet it underscores the promising antagonistic potential of any of the three T. harzianum strains against F. verticillioides. More work however is needed to be done to ascertain the mode(s) of inhibition. In the experiments of Sobowale et al. (2007), performance of T. harzianum strain 2 against F. verticillioides within maize (Z. mays) stem in the field also showed a promising antagonistic capability of this Trichoderma strain in vivo.

The agar plates which were not coloured at any culture point in the whole experiment might suggest the lack of metabolite production by the T. harzianum strains. However the clear zone which was observed only in 'simultaneous inoculation of pathogen and antagonist' for T. harzianum strains 1 and 2 might suggest a probable production of colourless metabolite by the two strains which moved ahead of them, colonizing space within the agar, thereby restricting further growth of F. verticillioides. Thrane et al. (2000) suggested that production of extracellular hydrolytic enzymes by Trichoderma species are important determinants of their antagonistic ability. However, this is not enough to suggest antibiosis as one of the modes of antagonism by the T. harzianum strains.

The lack of significance amongst the three T. harzianum strains in their growth inhibition of F. verticillioides suggests closeness in antagonistic potential of the three Trichoderma strains against F. verticillioides (R2 = 0.96). Any of the three Trichoderma strains could then be said to be good enough to successfully inhibit growth of F. verticillioides. Significance of 'inoculating antagonist before pathogen' over other two pairing methods, even at p = 0.01 is indicative of its preference over them, for effective growth inhibition of F. vertcillioides. This means that for effective growth inhibition of F. verticillioides, it is better to have any of the three T. harzianum strains inoculated before F. verticillioides. Deductively, this also suggests, that if any of the T. harzianum strains is tried as potential antagonists of F. verticillioides in vivo, it may be better for the antagonists to colonize tissues of the maize (Z. mays) plant ahead of F. verticillioides for a more effective growth suppression of the latter within maize plant. This was true for T. harzianum strain 2 when tried against F. verticillioides within maize (Z. mays) stem in the field (Sobowale et al. 2007). Significance of 'simultaneous inoculation of pathogen and antagonist' over 'inoculating pathogen before antagonist' at p = 0.05 suggests that it is even better for the T. harzianum strains to be inoculated simultaneously with F. verticillioides than for pathogen to be inoculated before

antagonist. This also suggests that for effective growth suppression of F. verticillioides within maize plant, it is better for any of the T. harzianum strains to occur at same time with the pathogen within tissues of the plant, than for the pathogen to occur before the antagonists.

A highly significant F value (p > 0.0001) for pairing method (Table 3) showed that growth inhibition of F. ver-ticillioides by the T. harzianum strains differed among pairing methods. This means that pairing method has an effect on effectiveness of growth inhibition of F. verticillioides by the T. harzianum strains. This confirms results shown in Table 2 obtained for the different pairing methods. The time of occurrence could therefore be said to play a significant role in determining indices of antagonism by the T. harzianum strains. This might be connected with their mode of antagonism which might have included competition for space and nutrients.

A significant F value (p > 0.026) for interaction between antagonist and pairing method showed that growth inhibition of F. verticillioides by any particular strain of T. har-zianum differed from one pairing method to the other. For instance, T. harzianum strain 1 differed significantly from strains 2 and 3 in its growth inhibition of F. verticillioides when pathogen was inoculated before antagonist (Table 4). It was however not significantly different in the other two pairing methods (Tables 5, 7). Growth inhibition of F. verticillioides by the T. harzianum strains could thus be said to be different among and within pairing methods. Significance of T. harzianum strain 1 over strains 2 and 3 is suggestive, particularly if the three of them are to be tried as potential antagonists of F. verticillioides within maize plant (Table 4).

In conclusion, it can be said that T. harzianum strain 1, with some more research, could address the problem of endophytic F. verticillioides within maize plant. The three Trichoderma strains might ultimately succeed as antagonists of F. verticillioides in vitro and in vivo, with some more research. If this is achieved, either by strain 1 or any of the other two strains of T. harzianum, incidence of fumonisins within maize seeds could also be a secondary target.

ACKNOWLEDGEMENTS

The research was sponsored by the International Institute of Tropical Agriculture, Ibadan, Nigeria. The authors also appreciate the efforts of Mr Lekan Ayinde, Mr Greg Ogbe and other staff of the Pathology Unit, IITA, Ibadan, Nigeria.

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POLISH SUMMARY

OGRANICZENIE WZROSTU FUSARIUM VERTICILLIOIDES NIREN. PRZY WYKORZYSTANIU SZCZEPOW TRICHODERMA HARZIANUM Z CZÇSCI ROSLIN KUKURYDZY (ZEA MAYS) I ICH RIZOSFERY

Porownano trzy szczepy Trichoderma harzianum (szczep 1: IMI 380934; szczep 2: IMI 380935 i szczep 3: IMI 380938) pod wzglçdem ich zdolnosci ograniczania odsrodkowe-go wzrostu Fusarium verticillioides in vitro. Kazdy szczep Trichoderma z zaszczepiono na pfytki Petriego o srednicy 9 cm, z zakwaszon^ pozywk^ agarowo-ziemniaczanq, stosuj^c 3 metody. Stosuj^c Procedure GLM SAS uzy-skano zroznicowane ograniczenie wzrostu patogena przez szczepy Trichoderma. Inhibicja wzrostu prze kazdy przez kazdy ze szczepow T. harzianum roznila siç istot-nie od kontroli, niezaleznie od wykorzystanej metody (p = 0,01, R2 = 0,96). Wyzsz^ inhibicjç F. verticillioides uzy-skano w przypadku wczesniejszego zaszczepienia anta-gonisty niz patogena, nawet przy p = 0,01. Mechanizm ograniczenia wzrostu obejmowal mykopasozytnictwo oraz konkurencjç o miejsce i skladniki pokarmowe. Inhibicja wzrostu patogena byla rozna pomiçdzy (p > 0.0001) i w ramach (p > 0.026) zastosowanych metod. T. harzianum szczep 1 lepiej ograniczal wzrost patogena niz dwa pozostale szczepy wtedy, gdy byl zaszczepiony wczesniej niz patogen, podczas gdy T. harzianum szczep 3 okazal siç lepszy, gdy patogena i antagonist^ zaszczepiono jed-noczesnie (p = 0,005). Wobec powyzszego rozne szczepy T. harzianum mog^, bye wykorzystywane jako obiecuj^cy antagonisci grzyba F. verticillioides.