Abstract
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
Introduction
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
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.
References
Altmore C, Norvell W A, Bjorkman T and Harman G E. 1999. solubilization of phosphates and micronutrients by the plant growth promoting and biocontrol fungus trichoderma harzianum rifai 1295-22. appl envi microbiol 65: 2926-2933.
Bevivino A, Tabacchioni C D S and Chiarini 2000. Efficacy of Burkholderia cepacia MCI 7 in disease suppression and growth promotion of maize. Biol Fert Soils 31: 225-231.
Biswas K K and Sen C. 2000. Management of stem rot of groundnut caused by Sclerotium rolfsii through Trichoderma harzianum. Indian Phytopath 53 (3): 290 – 295.
Dubey S C, Suresh M and Singh B. 2007. Evaluation of Trichoderma species against Fusarium oxysporum f. sp. ciceris for integrated management of chickpea wilt. Biol Contl 40: 118-127.
Elad Y. 2000. Biological control of foliar pathogens by means of T.harzianum and potential modes of action. Crop Prot 19: 709-714.
Hasan H A H. 2002. Gibberellin and auxin-indole production by plant root-fung and their biosynthesis under salinity-calcium interaction. Rostlinna Vyroba 48 (3): 101-106.
Hyakumachi M. 1994. Plant–growth–promoting fungi from turfgrass hizosphere with potential for disease suppression. Soil Microorganisms 44: 53-68.
Hyakumachi M. 2000. mehanisms of plant growth promotion by pgpf. in: fungal biotechnology in agricultural, food, and environmental applications, mycology, arora d. k. 21: 103.
Hyakumachi M. 2000. studies on biological control of soil borne plant pathogens. j gen pl pathol 66: 272-274.
Kang S C, Ha C G, Lee T G And Maheshwari D K. 2002. solubilization of insoluble inorganic phosphates by a soil inhabiting fungus fomitopsis sp. ps 102. cur sci 82: no. 4.
Noveriza R and Quimio T H. 2004. Soil mycoflora of black pepper rhizosphere in the Philippines and their in vitro antagonism against Phytophthora capsici. Indonesian J Agric Sci 5 (1): 1–10.
Shivanna M B, Meera M S and Hyakumachi M. 1994. Sterile fungi from zoysiagrass rhizosphere as plant growth promoters in spring wheat. Can J Microbiol 40: 637-644.
Warcup, J. H. 1950. The soil plate method for isolation of fungi from soil. Nature, Lond., 166 : 117.
