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Management of Fusarium Wilt of Tomato by Soil Amendment with Trichoderma koningii and a white sterile fungus

Management of Fusarium Wilt of Tomato by Soil Amendment with Trichoderma koningii and a white sterile fungus


Department of Botany, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India, E-mail: ________________________________________________________________________

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Fusarium wilt of tomato continues to incurr yield losses at different locations where it is endemic. The growth promoting ability of Trichoderma koningii  and a white sterile fungus (which did not fructify) was tested on tomato plants grown in soil inoculated with wilt pathogen Fusarium oxysporum f. sp. lycopersici. Tomato grown in the soil amended with combined inocula of the individual plant growth promoting fungi (PGPF) and wilt pathogen showed much a reduced intensity of the disease and promoted growth and yield. The antagonistic rhizosphere PGPF suppressed the deleterious soil microbes by competing at the active sites, reduced the intensity of disease development and, subsequently, stimulated the growth and yield of plants. These fungi may be used to suppress the wilt pathogen and raise the yield of tomato.

Key words: Fusarium wilt, Trichodermakoningii and tomato yield



Fusarium wilt of tomato caused by the fungus Fusarium oxysporum f. sp. lycopersici causes great loss in warm climates and sandy soils of temperate regions. Several chemical fungicides such as Bavistin etc. are used to suppress the disease but these chemicals have a negative impact on human health and are hazardous to the environment. A better alternative to chemicals are the soil microbes such as Trichoderma, Penicillium, etc. residing in the rhizosphere of crop plants that have the  ability to suppress the pathogens (Hyakumachi et al. 1994) and stimulate plant growth by the production of phytohormones (Hasan, 2002) and/or degradation of complex substrates (Altmore et al. 1999). Antagonistic nature of T. virens and Aspergillus against Phytophthora capsici causing foot root disease of black pepper has been reported (Noveriza et al. 2004). Metabolites of T. harzianum. T. viride and T.virens have been found to inhibit the mycelial growth of Fusarium oxysporum f. sp. ciceri causing wilt disease of chick pea (Dubey et al. 2007). Burkholderia cepacia MCI 7 has been reported as a promising inoculant for maize in the soil infested with a pathogenic strain of Fusarium moniliforme (Bevivino et al. 2000). The indigenous soil PGPF has been reported to suppress pathogenic Pythium spp. and stimulate the growth and yield of cucumber (Hyakumachi, 1994). Aspergillus niger, A. flavus, Penicillium corylophilum, P. cyclopium, P. funiculosum and Rhizopus stolonifer have been reported to produce gibberellin which is a growth regulating hormone in higher plants (Hasan, 2002). Altmore et al. (1999) investigated the capability of Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize some insoluble or sparingly soluble minerals in vitro and reported that T-22 was able to solubilize MnO2, metallic zinc and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium. 


Materials and Methods

Isolation of wilt pathogen, Trichoderma koningii  and a white sterile fungus from rhizosphere

Tomato plants were collected at regular growth intervals i.e. seedling, vegetative, flowering and fruiting stages from a field at Varanasi (Karaudi), India. Care was taken to dig out, as far as possible, the whole root system with a sterilized spatula. The root systems were then brought to the laboratory in separate polyethylene bags. The roots were given gentle taping to loosen-off the lightly adhering soil, in order to have just the rhizosphere soil attached to the root system. Small pieces of roots (2 cm) of different diam. were cut with sterilized scissors under aseptic condition and 25 such root pieces for each sample were transferred to flasks (one for healthy and the other for diseased roots) containing 100 ml of sterilized distilled water. The flasks were shaken vigorously with the help of a shaker to get a homogenous suspension of the rhizosphere soil. Taking this as the stock solution, conventional soil dilution plate method (Warcup, 1950) was followed for isolation of the rhizosphere fungi. Dilutions of 1:100, 1:1000, and 1:10000 were prepared. Three replicates of sterilized Petri plates were inoculated with one ml aliquots from all the diluted suspensions. To this was added 20 ml melted and cooled (40 C) potato dextrose agar medium and the plates were rotated slowly in clock-wise and anti-clock wise directions to disperse the soil solution uniformly in the culture medium. All the inoculated plates were then incubated at 25±2 C. The plates were examined regularly and the colonies of fungi appearing on the medium were transferred into fresh sterilized Petri plates containing PDA medium to avoid over-running by the fast growing forms. The pure cultures of the fungi thus isolated were preserved on PDA slants at 4C.

Preparation of mass culture

The mass culture of the rhizosphere fungi was prepared on barley grains (Shivanna et al. 1994). Clean and intact barley grains were taken for this purpose. The grains were pre-wetted by boiling them in water for 20-30 min so as to raise the moisture content of the grains up to 40-50% and to make them soft enough for the profuse growth of the fungus. After boiling, the grains were spread on wire mesh so as to drain the excess of water. The grains were then mixed with gypsum (calcium sulphate 2%) and chalk powder (calcium carbonate 0.5%) on dry weight basis to check pH of the medium and prevent grains from sticking with each other. Clean glucose bottles were filled with such barley grains (100g each) which were then steam sterilized for 1-2 h. The bottles were then allowed to cool at room temp. and inoculated with five agar blocks (5 mm diam. each) cut from the margin of actively growing culture of each fungus. The bottles were incubated at 25 ± 2C for 10 d. The bottles were shaken once or twice daily for rapid and uniform colonization of the fungi. Barley grains colonized by the rhizosphere fungi were air dried and aseptically stored at 4C for further use.

Preparation of pots

The soil sample was collected from the agricultural field at Varanasi, India. The soil was air dried at room temperature and ground to fine powder form with the help of pestle and mortar. The pure inoculum of each rhizosphere fungus, which was prepared on barley grains, was mixed separately with sterilized natural soil (1% w/w). The pure inoculum of the test pathogen (FOL) prepared separately on barley grains was mixed with each sample of sterilized natural soil inoculated with pure inoculum of individual rhizosphere fungi (1% w/w). The soil samples so prepared were separately filled in clay pots (15 ´ 25 cm). The pots were kept at room temperature for a week during which rhizosphere fungi and the test pathogen developed and colonized the soil particles. Soil supplemented with barley grains without inocula was used as control. The moisture level of the soil (25-30%) was maintained by watering the pots from time to time. Twenty surface sterilized seeds of variety H-24 of tomato were sown in each pot 8d after combined soil amendment with rhizosphere fungus and wilt pathogen. The experiments were set in replicates of three pots in a greenhouse. The observations for the combined effect of each rhizosphere fungus and wilt pathogen on growth and yield of tomato plants were made on plant height, dry weight of plant, fruits/plant, and weight of 100 dry seeds at 30, 50, 80, and 100 d after sowing (DAS). Ten plants were uprooted randomly from each treatment and the plant length above the ground was measured in cm and average height per plant was calculated. The same plants used for plant height were oven dried for 48 hours and average dry weight per plant was calculated. Fruits on all the tomato plants when formed and developed under each treatment were counted and average number of fruits per plant was calculated. The seeds separated out from the ripened fruits separately in each treatment were air dried. One hundred dry seeds were randomly selected from triplicate sets from individual treatments and were weighed.


Results and Discussion


The results clearly indicated that Trichoderma koningii and white sterile fungus promoted the growth of tomato in presence of wilt pathogen FOL (Cf. control). Out of these, T. koningii was found to be the maximum growth promoter at 30 DAS followed by white sterile fungus. Plant growth promotion by these fungi was significantly higher than the control (P < 0.05). Growth promotion of tomato increased with time up to 110 DAS. In case of FOL treatment plant growth was significantly lower at 30 DAS (Cf. control).

      Dry weight of plants was also significantly higher in PGPF treatments as compared to control (P < 0.05) and increased with time up to 110 DAS. In FOL treatment, dry weight of plants was considerably poor at 110 DAS (Cf. control). Maximum number of fruits/plant were recorded at 110 DAS with T. koningii treatment that was followed by white sterile fungus which was significantly higher than the control (P < 0.05). In FOL treatment, no fruit setting was observed. Weight of 100 dry seeds (observed at 110DAS) was maximum in T. koningii treatment which was followed by white sterile fungus which was significantly higher than the control (P < 0.05). In FOL treatment weight of dry seeds was not observed because of no fruit setting.

          Plant growth stimulation by the rhizosphere PGPF can be attributed to their ability to suppress pathogenic soil microorganisms, produce growth promoting substances such as phytohormones and/or degrade complex substrates in the soil. Because of their antagonistic nature, the PGPF might have suppressed the wilt pathogen FOL by competing at the active sites (Biswas and Sen, 2000). This way, the test pathogen would not have infected the tomato plants and subsequently the intensity of disease development might have been low or negligible (Hyakumachi, 1994). Elad (2000) studied the biological control of foliar pathogens of cucumber by means of T. harzianum and found that 4 foliar pathogens namely Botrytis cinerea, Pseuperonospora cubensis, Sclerotinia sclerotiorum and Sphaerotheca fusca causing grey mould, downy mildew, white mould and powdery mildew diseases of cucumber, respectively, were suppressed by T. harzianum under greenhouse conditions. Narisawa et al. (2004) reported that Verticillium dahliae causing wilt disease of eggplant was suppressed by Heterconium chaetospira, Phialocephala fortinii, Penicillium sp. and Trichoderma sp. T. harzianum, T. viride and T. virens have been found to suppress the mycelial growth of Fusarium oxysporum f. sp. ciceris and enhance the growth and yield of this crop plant (Dubey et al. 2007).

PGPF have been reported to mineralize the organic substrates and may, therefore, provide the plants with necessary mineral nutrients in an easily assimilating form (Hyakumachi, 2000). Altmore et al. (1999) investigated the capability of Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize some insoluble or sparingly soluble minerals in vitro and reported that T-22 was able to solubilize MnO2, metallic zinc and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium. This phosphate solubilising activity of T. harzianum might be responsible for its plant growth promoting ability. Kang et al. (2002) reported the ability of Fomitopsis to solubilize tri-calcium phosphate. 



Table. Effect of soil amendment with Trichoderma koningii  and a white sterile fungus in presence of wilt pathogen on growth and yield of tomato (in pots)



     PGPF            No. of       Plant              Dry weight      No. of      Weight of

                          days        height            of plant (g)      fruits/      100 dry

                                          (cm)                                      plant*      seeds (g) **



Trichoderma        30        9.0 ± 0.05       0.67 ± 0.00              -                 -

koningii                 50    13.06 ± 0.01         1.1 ± 0.05              -                 -

                             80    26.06 ± 0 06       1.27 ± 0.00     6 ± 0.0                 -

                           110    30.06 ± 0.03      1.75 ±  0.00   12 ± 0.0      0.79 ± 0



White sterile        30        8.1 ± 0.05         0.65 ± 0.04              -                 -

Fungus                50     13.36 ± 0.08        1.02 ± 0.02               -                 - 

                             80      27.2 ± 0.05         1.32 ± 0.00      5 ± 0.8                 -

                           110    30.03 ± 0.03         1.56 ± 0.00      9 ± 0.8   0.64 ± 0.0


FOL                      30     3.46 ± 0.03         0.31 ± 0.00               -                  -            

                              50       6.1 ± 0.05         0.41 ± 0.00                -                  -    

                               80   10.36 ± 0.08         0.97 ± 0.00                -                  -

                             110   12.13 ± 0.08         0.97 ± 0.00                -                  -


Control                   30       6.7 ± 0.30          0.54 ± 0.08                -                  -

 (without                50     12.0 ± 0.10            1.0 ± 0.10                -                  -   

 PGPF and            80     25.0 ± 0.20          1.26 ± 0.02       1 ± 0.3                 -

    FOL)                110     29.5 ± 0.30          1.50 ± 0.08       4 ± 0.8   0.40 ± 0.0    



Average of all three replicates; ±, Standard error of mean (SEM). 

Data were statistically analyzed which were found to be significant (P < 0.05)

-, Not recorded *Yield in terms of number of fruits only.






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