Abstract: Trichoderma viride, T. polysporum, T. hamatum and T. aureoviride were used as potential biological agents for seed treatments against Ceratocystis paradoxa causing black seed rot in oil palm sprouted seeds in vitro. T. polysporum significantly reduced the percentage infection to 13 and 10%, 4 days post inoculation. T. viride 18.3 and 11.7%, T. hamatum 22 and 15%, T. aureoviride 23.7 and 13%, benlate solution 21.7 and 18.3% and control 80 and 30% for wounded and unwounded seeds, respectively. In vivo, the emergence (survival) and the growth of oil palm seedlings from Trichoderma species treated oil palm sprouted seeds were significantly higher than the emergence and the growth observed from the control treatment. C. paradoxa infested seeds had 63 to 71% survival and average seedling heights of 18.4 to 25.1 cm. Seeds without pathogen had 100% survival with average seedling heights of 38.2 to 46.5 cm. The effects of the bioagents were similar or high than the one obtained from benlate solution.
INTRODUCTION
The oil palm (Elaeis guineensis Jacq.) is said to be indigenous to West Africa (Zeven, 1965; Corner, 1966). The oil palm industry provides direct employment to about four million Nigerian people in about twenty oil palm growing states in Nigeria and indirectly to other numerous people involved in processing and marketing (Ahmed, 2001).
Despite the enormous potential of the oil palm, there is problem with soil borne fungus, Ceratocystis paradoxa causing black seed rot disease in oil palm sprouted seeds. The cause of dry basal rot in adult palm, conducted by Robertson (1962) was shown to be due to the Ascomycete, C. paradoxa of which the imperfect stage is known as Thielaviopsis paradoxa. It is a soil inhabitant, widely distributed throughout the tropics of Africa and Asia and causes disease of several other crops (Rajagopalan, 1965).
An outbreak of C. paradoxa in 1982 caused black rot of plumule and radicle of sprouted seeds in a seed store at Nigerian Institute for Oil Palm Research (NIFOR). About 109,000 sprouted seeds (72.7%) of the total stock were affected resulting in a huge financial loss to the institute (Omamor, 1985). Although, binomial is recommended for the treatment of C. paradoxa causing seed rot of sprouted seeds of the oil palm (Omamor, 1985). However, fungicide seed treatment is not desirable for disease control due to some adverse effects on the environmental and ecosystem, such as harm to non target organisms, animals and plants. This results in residues in soil and contamination of the water and food chains. Fungicides may induce pathogen resistance, making their effects variable and short lived (Cook and Baker, 1983). In addition, fungicides are expensive in comparison with the relatively low commodity price of sprouted seeds of the oil palm. Hence, there is a need for an improved control management system with reduced fungicide use.
Biological control of plant pathogens by microorganisms has been considered a more natural and environmentally acceptable alternative to the existing chemical treatment methods (Baker and Paulilitz, 1996; Cook and Baker, 1983). Harman et al. (1980) and Nelson et al. (1988) reported the use of Trichoderma hamatum for the control of Pythium seed rot and Rhizoctonia root rot in pea. Hwang and Chakravarty (1992, 1993) reported findings of Bacillus subtilis and Gliocladium virens (syn. Trichoderma virens) as bioagents against Rhizoctonia root rot when employed with a fungicide.
Since the bioagents are effective and has been pursued against seed and root rot pathogens with success, biological control against C. paradoxa causing seed rot of sprouted oil palm seeds has not been pursued. The objective of the present study was to examine the efficacy of Trichoderma species on oil palm sprouted seeds for the control of C. paradoxa and compare with commercial benlate fungicide.
MATERIALS AND METHODS
Sprouted seeds of the oil palm used in these investigations were raised from germinated improved seed (Tenera hybrid) produced at the Nigerian Institute for Oil Palm Research (NIFOR) near Benin City, Edo State. The seeds were sprouted by the heat treatment method (Anonymous, 1965). Before the seeds were sprouted, they were soaked in 90% (vol./vol.) ethanol for 2 min and washed five times with sterile distilled water. Treated seeds were transferred to sterile filter paper to absorb excess water and then disinfected in 2% (wt./vol.) sodium hypochlorite (Sigma-Aldrich Chenire) for 2 min (Yedidia et al., 1999). The disinfected seeds were washed five times in sterile distilled water and excess water was removed with sterilized filter paper under asceptic conditions and left in sterile polythene bags for sprouting. The studies were conducted in 2005 under glasshouse conditions at botanical garden and botany and microbiology laboratories, University of Lagos, Nigeria.
A total number of 720 oil palm sprouted seeds were used for in-vitro seed treatments, 360 wounded (stapped with sterile inoculating needle) and 360 unwounded sprouted seeds, all showing presence of radicles and plumules. One hundred and twenty seeds of which 60 wounded and 60 unwounded (20 wounded and 20 unwounded replicated 3 times) were used for each treatment. The fungal cultures used in this investigation were previously isolated by the authors from diseased sprouted seeds of the oil palm collected from the above-mentioned location (NIFOR) in 2004. They are Trichoderma viride, T. hamatum, T. polysporum, T. aureoviride and the pathogenic fungus Ceratocystis paradoxa, the causal agent of black seed rot of sprouted oil palm seeds.
Sprouted seeds, selected for the uniformity (length of radicle and plumule) were placed in each Trichoderma species suspension (25x106). For the benlate solution (1 g 100 mL), sprouted seeds were soaked for 10 min. They were incubated for 24 h. The sprouted seeds were challenged by (radicle and plumule) root dipping in a conidia suspension of C. paradoxa (25x106). Non-challenged control sprouted seeds were dipped in sterile distilled water in the same condition. The treatment were as follows:
| • | Trichoderma sp. or benlate/C. paradoxa |
| • | Control/sterile distilled water |
They were maintained in sterile petri dish plates and incubated at 28o C. Percentage infection was determined on 1, 3, 7 and 14 days post inoculation.
In vivo oil palm sprouted seed treatments: A total number of 864 oil palm sprouted seeds (432 C. paradoxa infested seeds and 432 seeds without pathogen) were used in glasshouse. Twenty four seeds replicated 3 times were used for each treatment. The sprouted seeds were planted with their plumules the right way up at a depth of 2.54 cm in black polythene bags filled with top soil (pH 5.5 to 6.5). Soil was sterilized at 121oC for 1 h for two successive days to destroy weeds, seeds, pests and microorganisms. Each sprouted seed was inoculated using sterile syringes with the same spore suspension and soak method of in-vitro earlier mentioned. The polythene bags had holes at their bottoms to allow excess water to drain away. The control experiment had sterile distilled water in the place of fungal cultures or benlate solution. The treatments were as follows:
| • | Trichoderma sp. or benlate/C. paradoxa |
| • | Trichoderma sp. or benlate/sterile water |
| • | Control/sterile distilled water |
The seedlings were watered as at when required. Oil palm seedlings heights and survivals were taken six months after planting. Both the in vitro and in vivo experiments were repeated twice.
RESULTS
Sprouted seeds of the oil palm treated with Trichoderma species and benlate solution reduced infection of Ceratocystis paradoxa, but the reduction varied. There were no infections recorded on the 1st day post inoculation. Infection was noticed on the 3rd day with Trichoderma polysporum, T. viride and benlate having significant (p<0.005) reductions than the other Trichoderma species (Table 1). On the 7th day T. polysporum, T. viride and benlate were significantly (p<0.005) effective and similar in reduction of C. paradoxa. But more consistence was T. polysporum with 13.0 and 10.0% for wounded and unwounded sprouted seeds on the 14th day.
| Table 1: | Effects of Trichoderma species and benlate solution
on sprouted seeds of the oil palmagainst Ceratocystis paradoxa |
*Mean values followed by are significant (p<0.05) according
to one-way analysis of variance as compared to the control. Values are average
of three replicates |
|
| Table 2: | Effects of Trichoderma species and benlate solution
on sprouted seeds of the oil palm against Ceratocystis paradoxa 6
months after planting in glasshouse conditions |
*Mean values followed by are significant (p<0.05) according
to one-way analysis of variance as compared to the control. Values are average
of three replicates |
|
However, the consistence of benlate dropped having 21.7 and 18.3% also on the 14th day (Table 1). Effect of T. polysporum was more significant compared with the control treatment (80 and 30%) but not much significant different from other Trichoderma species and benlate.
Trichoderma species and benlate solution treatments on sprouted seeds of the oil palm increased emergence (percentage of survival) and reduced percentage of infection (Table 2). Both emergence and the growth (height) of oil palm seedlings from antagonists treated seeds were significantly (p<0.005) higher than the emergence and the growth observed from C. paradoxa infested seeds and the control. T. viride and T. polysporum increased emergence and growth of oil palm seedlings by 71% and 23.8 cm and 71% and 21.2 cm, respectively (Table 2). These are not significantly different from the one obtained from benlate (71% and 25.1 cm). The effects of reductions were significantly higher when compared with the control but not significantly different among Trichoderma species.
DISCUSSION
Trichoderma sp. are active ingredients in a variety of commercial biofungicides used to control a range of economically important aerial and soil borne fungal plant pathogens (Harman, 2000; Harman and Kubicek, 1998). The observations that Trichoderma species applied to sprouted seeds of the oil palm significantly reduced infection of C. paradoxa suggested that the bioagents have the substantial potential to control many soilborne fungal pathogens involved in seed decay. However, the reason for the dropped in consistence of benlate solution on the 14th day post inoculation (Table 1) was not given in this study.
The emergence and height of oil palm seedlings from antagonists treated seeds (Table 2), suggested active growth. This active growth may have been supported by the production of extracellular enzymes capable of releasing cell wall components that provide nutrients and/or further stimulated host colonization (Lorito and Woo, 1998; Woo et al., 2000).
The unwounded (new developing radicle and plumule) sprouted seeds were more susceptible while the older ones (with ticker radicle and plumule) were resistant to attack by C. paradoxa. Infection was high with wounded sprouted seeds when compared with the unwounded. C. paradoxa has been reported as pineapple disease of sugarcane setts, entering the seed piece through cut ends, destroys the buds, thus causing germination failure (Yin and Hoy, 1997, 1998). Although germination failure was observed more with control treatments, treated with C. paradoxa infested seeds while seeds treated with Trichoderma species had significant reduction in germination failure.
The results of this investigation have confirmed the efficacy of Trichoderma species to control C. paradoxa with similar or high effects when compared with benlate fungicide. Because of the remarkable properties and potentials of Trichoderma species, more studies of these bioagents should be encouraged.
ACKNOWLEDGMENT
I wish to thank the Director, Board and Management members of Nigerian Institute for Oil Palm Research (NIFOR) for the award of scholarship and provision of oil palm sprouted seeds.